Rna interference mediated inhibition of adenosine a1 receptor (adora1) gene expression using short interfering rna

ABSTRACT

The present invention concerns methods and reagents useful in modulating adenosine A1 receptor (ADORA1) gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to small interfering RNA (siRNA) molecules capable of mediating RNA interference (RNAi) against ADORA1 and related receptors.

This application is a continuation application of U.S. patentapplication Ser. No. 10/224,005, filed Aug. 20, 2002, which claims thebenefit of U.S. Provisional Application No. 60/315,315 filed Aug. 28,2001, U.S. Provisional Application No. 60/350,580, filed Feb. 20, 2002,U.S. Provisional Application No. 60/363,124, filed Mar. 11, 2002, andU.S. Provisional Application No. 60/386,782, filed Jun. 6, 2002, all ofwhich are herein incorporated by reference in their entireties,including the drawings.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR§1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “SequenceListing9USCNT”,created on Aug. 28, 2008, which is 87,964 bytes in size.

BACKGROUND OF THE INVENTION

The present invention concerns methods and reagents useful in modulatinggene expression associated with asthma, inflammation and allergicresponse in a variety of applications, including use in therapeutic,diagnostic, target validation, and genomic discovery applications.Specifically, the invention relates to short interfering nucleic acidmolecules (siRNA) capable of mediating RNA interference (RNAi) againstadenosine A1 receptor gene expression.

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention.

RNA interference refers to the process of sequence-specific posttranscriptional gene silencing in animals mediated by short interferingRNAs (siRNA) (Fire et al., 1998, Nature, 391, 806). The correspondingprocess in plants is commonly referred to as post transcriptional genesilencing or RNA silencing and is also referred to as quelling in fungi.The process of post transcriptional gene silencing is thought to be anevolutionarily conserved cellular defense mechanism used to prevent theexpression of foreign genes which is commonly shared by diverse floraand phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protectionfrom foreign gene expression may have evolved in response to theproduction of double stranded RNAs (dsRNA) derived from viral infectionor the random integration of transposon elements into a host genome viaa cellular response that specifically destroys homologous singlestranded RNA or viral genomic RNA. The presence of dsRNA in cellstriggers the RNAi response though a mechanism that has yet to be fullycharacterized. This mechanism appears to be different from theinterferon response that results from dsRNA mediated activation ofprotein kinase PKR and 2′,5′-oligoadenylate synthetase resulting innon-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21-23 nucleotides in length and comprise about 19 base pair duplexes.Dicer has also been implicated in the excision of 21 and 22 nucleotidesmall temporal RNAs (stRNA) from precursor RNA of conserved structurethat are implicated in translational control (Hutvagner et al., 2001,Science, 293, 834). The RNAi response also features an endonucleasecomplex containing a siRNA, commonly referred to as an RNA-inducedsilencing complex (RISC), which mediates cleavage of single stranded RNAhaving sequence complementary to the antisense strand of the siRNAduplex. Cleavage of the target RNA takes place in the middle of theregion complementary to the antisense strand of the siRNA duplex(Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates (Elbashir et al., 2001, EMBO J., 20, 6877) has revealed certainrequirements for siRNA length, structure, chemical composition, andsequence that are essential to mediate efficient RNAi activity. Thesestudies have shown that 21 nucleotide siRNA duplexes are most activewhen containing two nucleotide 3′-overhangs. Furthermore, completesubstitution of one or both siRNA strands with 2′-deoxy (2′-H) or2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution ofthe 3′-terminal siRNA overhang nucleotides with deoxy nucleotides (2′-H)was shown to be tolerated. Single mismatch sequences in the center ofthe siRNA duplex were also shown to abolish RNAi activity. In addition,these studies also indicate that the position of the cleavage site inthe target RNA is defined by the 5′-end of the siRNA guide sequencerather than the 3′-end (Elbashir et al., 2001, EMBO J., 20, 6877). Otherstudies have indicated that a 5′-phosphate on the target-complementarystrand of a siRNA duplex is required for siRNA activity and that ATP isutilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen etal., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-overhanging segments of a21-mer siRNA duplex having 2 nucleotide 3′ overhangs withdeoxyribonucleotides does not have an adverse effect on RNAi activity.Replacing up to 4 nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877). In addition,Elbashir et al., supra, also report that substitution of siRNA with2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al.,International PCT Publication No. WO 00/44914, and Beach et al.,International PCT Publication No. WO 01/68836 both suggest that siRNA“may include modifications to either the phosphate-sugar back bone orthe nucleoside to include at least one of a nitrogen or sulfurheteroatom”, however neither application teaches to what extent thesemodifications are tolerated in siRNA molecules nor provide any examplesof such modified siRNA. Kreutzer and Limmer, Canadian Patent ApplicationNo. 2,359,180, also describe certain chemical modifications for use indsRNA constructs in order to counteract activation of doublestranded-RNA-dependent protein kinase PKR, specifically 2′-amino or2′-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer and Limmer similarly fail to show towhat extent these modifications are tolerated in siRNA molecules nor dothey provide any examples of such modified siRNA.

Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that “RNAs with two [phosphorothioate] modified bases also hadsubstantial decreases in effectiveness as RNAi triggers (data notshown); [phosphorothioate] modification of more than two residuesgreatly destabilized the RNAs in vitro and we were not able to assayinterference activities.” Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and observed that substituting deoxynucleotides forribonucleotides “produced a substantial decrease in interferenceactivity”, especially in the case of Uridine to Thymidine and/orCytidine to deoxy-Cytidine substitutions. Id. In addition, the authorstested certain base modifications, including substituting 4-thiouracil,5-bromouracil, 5-iodouracil, 3-(aminoallyl)uracil for uracil, andinosine for guanosine in sense and antisense strands of the siRNA, andfound that whereas 4-thiouracil and 5-bromouracil were all welltolerated, inosine “produced a substantial decrease in interferenceactivity” when incorporated in either strand. Incorporation of5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resultedin substantial decrease in RNAi activity as well.

Beach et al., International PCT Publication No. WO 01/68836, describesspecific methods for attenuating gene expression using endogenouslyderived dsRNA. Tuschl et al., International PCT Publication No. WO01/75164, describes a Drosophila in vitro RNAi system and the use ofspecific siRNA molecules for certain functional genomic and certaintherapeutic applications; although Tuschl, 2001, Chem. Biochem., 2,239-245, doubts that RNAi can be used to cure genetic diseases or viralinfection due “to the danger of activating interferon response”. Li etal., International PCT Publication No. WO 00/44914, describes the use ofspecific dsRNAs for use in attenuating the expression of certain targetgenes. Zernicka-Goetz et al., International PCT Publication No. WO01/36646, describes certain methods for inhibiting the expression ofparticular genes in mammalian cells using certain dsRNA molecules. Fireet al., International PCT Publication No. WO 99/32619, describesparticular methods for introducing certain dsRNA molecules into cellsfor use in inhibiting gene expression. Plaetinck et al., InternationalPCT Publication No. WO 00/01846, describes certain methods foridentifying specific genes responsible for conferring a particularphenotype in a cell using specific dsRNA molecules. Mello et al.,International PCT Publication No. WO 01/29058, describes theidentification of specific genes involved in dsRNA mediated RNAi.Deschamps Depaillette et al., International PCT Publication No. WO99/07409, describes specific compositions consisting of particular dsRNAmolecules combined with certain anti-viral agents. Waterhouse et al.,International PCT Publication No. 99/53050, describes certain methodsfor decreasing the phenotypic expression of a nucleic acid in plantcells. Driscoll et al., International PCT Publication No. WO 01/49844,describes specific DNA constructs for use in facilitating gene silencingin targeted organisms. Parrish et al., 2000, Molecular Cell, 6,1977-1087, describes specific chemically modified siRNA constructstargeting the unc-22 gene of C. elegans. Grossniklaus, International PCTPublication No. WO 01/38551, describes certain methods for regulatingpolycomb gene expression in plants. Churikov et al., International PCTPublication No. WO 01/42443, describes certain methods for modifyinggenetic characteristics of an organism. Cogoni et al., International PCTPublication No. WO 01/53475, describes certain methods for isolating aNeurospora silencing gene and uses thereof Reed et al., InternationalPCT Publication No. WO 01/68836, describes certain methods for genesilencing in plants. Honer et al., International PCT Publication No. WO01/70944, describes certain methods of drug screening using transgenicnematodes as Parkinson's disease models. Deak et al., International PCTPublication No. WO 01/72774, describes certain Drosophila derived geneproducts. Arndt et al., International PCT Publication No. WO 01/92513describes certain methods for mediating gene suppression by usingfactors that enhance RNAi. Tuschl et al., International PCT PublicationNo. WO 02/44321, describe certain synthetic siRNA constructs. Pachuk etal., International PCT Publication No. WO 00/63364, and Satishchandranet al., International PCT Publication No. WO 01/04313 describes certainmethods and compositions for inhibiting the function of certainpolynucleotide sequences. Echeverri et al., International PCTPublication No. WO 02/38805, describes certain C elegans genesidentified via RNAi. Kreutzer et al., International PCT Publication No.WO 02/055692 and WO 02/055693, describes certain methods for inhibitinggene expression using RNAi.

Asthma is a chronic inflammatory disorder of the lungs characterized byairflow obstruction, bronchial hyper-responsiveness, and airwayinflammation. T-lymphocytes that produce T_(H)2 cytokines andeosinophilic leukocytes infiltrate the airways. In the airway and inbronchial alveolar lavage (BAL) fluid of individuals with asthma, highconcentrations of T_(H)2 cytokines, interleukin-4 (IL-4), IL-5, andIL-13, are present along with increased levels of adenosine. In contrastto normal individuals, asthmatics respond to adenosine challenge withmarked airway obstruction. Upon allergen challenge, mast cells areactivated by cross-linked IgE-allergen complexes. Large amounts ofprostaglandin D2 (PGD2), the major cyclooxygenase product of arachidonicacid are released. PGD2 is generated from PGH2 via the activity ofprostaglandin D2 synthetase (PTGDS). PGD2 receptors and adenosine A1receptors are present in the lungs and airway along with various othertissues in response to allergic stimuli (Howarth, 1997, Allergy, 52,12).

The significance of PGD2 as a mediator of allergic asthma has beenestablished with the development of mice deficient in the PGD2 receptor(DP). DP is a heterotrimeric GTP-binding protein-coupled, rhodopsin-typereceptor specific for PGD2 (Hirata et al., 1994, PNAS USA., 91, 11192).These mice fail to develop airway hyperreactivity and have greatlyreduced eosinophil infiltration and cytokine accumulation in response toallergens. Upon allergen challenge mice deficient in the prostaglandinD2 (PGD2) receptor (DP) did not develop airway hyperactivity. Cytokine,lymphocyte and eosinophil accumulation in the lungs were greatly reduced(Matsuoka et al., 2000, Science, 287, 2013). The DP −/− mice exhibitedno behavioral, anatomic, or histological abnormalities. Primary immuneresponse is not affected by DP disruption.

Asthma affects more than 100 million people worldwide and more than 17million Americans (5% of the population). Since 1980 the incidence hasmore than doubled and deaths have tripled (5,000 deaths in 1995). Annualasthma-related healthcare costs in the US alone were estimated to exceed$14.5 billion in 2000. Current therapies such as inhalantanti-inflammatories and bronchodilators can be used to treat symptoms,however, these therapies do not prevent or cure asthma.

SUMMARY OF THE INVENTION

One embodiment of the invention provides a short interfering RNA (siRNA)molecule that down regulates expression of adenosine A1 receptor(ADORA1) by RNA interference. The siRNA molecule can be adapted for useto treat, for example allergic/inflammatory diseases and conditions,including but not limited to asthma, allergic rhinitis, atopicdermatitis, and any other indications that can respond to the level ofADORA1. The siRNA molecule can comprise a sense region and an antisenseregion. The antisense region can comprise sequence complementary to anRNA sequence encoding ADORA1 and the sense region can comprise sequencecomplementary to the antisense region. An siRNA molecule of theinvention can be adapted for use to treat asthma.

An siRNA molecule can comprise a sense region and an antisense regionand wherein said antisense region comprises sequence complementary to anRNA sequence encoding ADORA1 and the sense region comprises sequencecomplementary to the antisense region.

The siRNA molecule can be assembled from two nucleic acid fragmentswherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of said siRNA molecule. The sense regionand antisense region can be covalently connected via a linker molecule.The linker molecule can be a polynucleotide linker or a non-nucleotidelinker.

The antisense region of ADORA1 siRNA constructs can comprise a sequencecomplementary to sequence having any of SEQ ID NOs. 1-161. The antisenseregion can also comprise sequence having any of SEQ ID NOs. 162-322,336, 338, 340, 342, 344, or 346. The sequences shown in SEQ ID NO:1-346are not limiting. A siRNA molecule of the invention can comprise anycontiguous ADORA1 sequences (e.g., about 19 contiguous ADORA1nucleotides. The sense region of ADORA1 siRNA constructs can comprisesequence having any of SEQ ID NOs. 1-161, 335, 337, 339, 341, 343, or345. The sense region can comprise a sequence of SEQ ID NO. 323 and theantisense region can comprise a sequence of SEQ ID NO. 324. The senseregion can comprise a sequence of SEQ ID NO. 325 and the antisenseregion can comprise a sequence of SEQ ID NO. 326. The sense region cancomprise a sequence of SEQ ID NO. 327 and the antisense region cancomprise a sequence of SEQ ID NO. 328. The sense region can comprise asequence of SEQ ID NO. 329 and the antisense region can comprise asequence of SEQ ID NO. 330. The sense region can comprise a sequence ofSEQ ID NO. 331 and the antisense region can comprise a sequence of SEQID NO. 332. The sense region can comprise a sequence of SEQ ID NO. 333and the antisense region can comprise a sequence of SEQ ID NO. 334.

The sense region of a siRNA molecule of the invention can comprise a3′-terminal overhang and the antisense region can comprise a 3′-terminaloverhang. The 3′-terminal overhangs each can comprise about 2nucleotides. The antisense region of the 3′-terminal nucleotide overhangcan be complementary to RNA encoding ADORA1.

The sense region of a siRNA molecule can comprise one or more (e.g.,about 1, 2, 3, 4, 5, or more) 2′-O-methyl modified pyrimidinenucleotides. The sense region can comprise a terminal cap moiety at the5′-end, 3′-end, or both 5′ and 3′ ends of said sense region.

The antisense region of a siRNA molecule can comprise one or more (e.g.,about 1, 2, 3, 4, 5, or more) 2′-deoxy-2′-fluoro modified pyrimidinenucleotides. The antisense region can also comprise a phosphorothioateinternucleotide linkage at the 3′ end of said antisense region. Theantisense region can comprise between about one and about fivephosphorothioate internucleotide linkages at the 5′ end of saidantisense region.

The 3′-terminal nucleotide overhangs of a siRNA molecule can compriseribonucleotides or deoxyribonucleotides that are chemically modified ata nucleic acid sugar, base, or backbone. The 3′-terminal nucleotideoverhangs can also comprise one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base ribonucleotides. Additionally, the 3′-terminalnucleotide overhangs can comprise one or more (e.g., about 1, 2, 3, 4,5, or more) acyclic nucleotides.

The 3′-terminal nucleotide overhangs can comprise nucleotides comprisinginternucleotide linkages having Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally occurring or chemicallymodified, each X and Y is independently O, S, N, alkyl, or substitutedalkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl,O-alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W, X, Y and Z are notall O.

The 3′-terminal nucleotide overhangs can comprise nucleotides ornon-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-OH, alkyl-SH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, 0-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula I; R9 is O,S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base or any othernon-naturally occurring base that can be complementary ornon-complementary to ADORA1 RNA or a non-nucleosidic base or any othernon-naturally occurring universal base that can be complementary ornon-complementary to ADORA1 RNA.

Another embodiment of the invention provides an expression vectorcomprising a nucleic acid sequence encoding at least one siRNA moleculeof the invention in a manner that allows expression of the nucleic acidmolecule. The expression vector can be in a mammalian cell, such as ahuman cell. The siRNA molecule can comprise a sense region and anantisense region. The antisense region can comprise sequencecomplementary to an RNA sequence encoding ADORA1 and the sense regioncomprises sequence complementary to the antisense region. The siRNAmolecule can comprise two distinct strands having complementarity senseand antisense regions or can comprise a single strand havingcomplementary sense and antisense regions.

Therefore, this invention relates to compounds, compositions, andmethods useful for modulating gene expression, for example, genesassociated with asthma, inflammation and allergic response by RNAinterference (RNAi) using short interfering RNA (siRNA). In particular,the instant invention features siRNA molecules and methods to modulatethe expression of ADORA1. The siRNA of the invention can be unmodifiedor chemically modified. The siRNA of the instant invention can bechemically synthesized, expressed from a vector or enzymaticallysynthesized. The instant invention also features various chemicallymodified synthetic short interfering RNA (siRNA) molecules capable ofmodulating ADORA1 gene expression/activity in cells by RNA inference(RNAi). The use of chemically modified siRNA is expected to improvevarious properties of native siRNA molecules through increasedresistance to nuclease degradation in vivo and/or improved cellularuptake. The siRNA molecules of the instant invention provide usefulreagents and methods for a variety of therapeutic, diagnostic,agricultural, target validation, genomic discovery, genetic engineeringand pharmacogenomic applications.

In one embodiment, the invention features one or more siRNA moleculesand methods that independently or in combination modulate the expressionof gene(s) encoding proteins associated with asthma, inflammation, andthe allergic response. Specifically, the present invention featuressiRNA molecules that modulate the expression of ADORA1 genes such asGenBank accession No. NM_(—)000674.

The description below of the various aspects and embodiments is providedwith reference to the exemplary gene ADORA1. However, the variousaspects and embodiments are also directed to other genes which expressother adenosine receptors (A2A, A2B, and/or A3). Those additional genescan be analyzed for target sites using the methods described for ADORA1.Thus, the inhibition and the effects of such inhibition of the othergenes can be performed as described herein. Thus, the inhibition and theeffects of such inhibition of the other genes can be performed asdescribed herein.

In one embodiment, the invention features a siRNA molecule that downregulates expression of an ADORA1 gene, for example, wherein the ADORA1gene comprises ADORA1 sequence.

In one embodiment, the invention features a siRNA molecule having RNAiactivity against ADORA1 RNA, wherein the siRNA molecule comprises asequence complimentary to any RNA having ADORA1 encoding sequence, suchas GenBank accession No. NM_(—)000674.

In another embodiment, the invention features a siRNA moleculecomprising sequences selected from the group consisting of SEQ ID NOs:1-322. In another embodiment, the invention features an ADORA1 siRNAmolecule having an antisense region complementary to any sequence havingSEQ ID NOs: 1-161. In another embodiment, the invention features anADORA1 siRNA molecule having an antisense region having any of SEQ IDNOs: 162-322, 336, 338, 340, 342, 344, 346, 348, 350, 352 or 354. Inanother embodiment, the invention features an ADORA1 siRNA moleculehaving a sense region having any of SEQ ID NOs. 1-161, 335, 337, 339,341, 343, or 345, 347, 349, 351 or 353. The sense region can comprise asequence of SEQ ID NO. 323 and the antisense region can comprise asequence of SEQ ID NO. 324. The sense region can comprise a sequence ofSEQ ID NO. 325 and the antisense region can comprise a sequence of SEQID NO. 326. The sense region can comprise a sequence of SEQ ID NO. 327and the antisense region can comprise a sequence of SEQ ID NO. 328. Thesense region can comprise a sequence of SEQ ID NO. 329 and the antisenseregion can comprise a sequence of SEQ ID NO. 330. The sense region cancomprise a sequence of SEQ ID NO. 331 and the antisense region cancomprise a sequence of SEQ ID NO. 332. The sense region can comprise asequence of SEQ ID NO. 333 and the antisense region can comprise asequence of SEQ ID NO. 334. In yet another embodiment, the inventionfeatures a siRNA molecule comprising a sequence, for example theantisense sequence of the siRNA construct, complementary to a sequenceor portion of sequence comprising GenBank accession No. NM_(—)000674.

In one embodiment, a siRNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by an ADORA1 gene.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response are doublestranded RNA molecules. In another embodiment, the siRNA molecules ofthe invention consist of duplexes containing about 19 base pairs betweenoligonucleotides comprising about 19 to about 25 nucleotides (e.g.,about 19, 20, 21, 22, 23, 24, or 25). In yet another embodiment, siRNAmolecules of the invention comprise duplexes with overhanging ends of1-3 (e.g., 1, 2, or 3) nucleotides, for example 21 nucleotide duplexeswith 19 base pairs and 2 nucleotide 3′-overhangs. These nucleotideoverhangs in the antisense strand are optionally complementary to thetarget sequence.

In one embodiment, the invention features chemically modified siRNAconstructs having specificity for ADORA1 expressing nucleic acidmolecules. Non-limiting examples of such chemical modifications includewithout limitation phosphorothioate internucleotide linkages, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides,“universal base” nucleotides, 5-C-methyl nucleotides, and inverteddeoxyabasic residue incorporation. These chemical modifications, whenused in various siRNA constructs, are shown to preserve RNAi activity incells while at the same time, dramatically increasing the serumstability of these compounds. Furthermore, contrary to the datapublished by Parrish et al., supra, applicant demonstrates that multiple(greater than one) phosphorothioate substitutions are well tolerated andconfer substantial increases in serum stability for modified siRNAconstructs. Chemical modifications of the siRNA constructs can also beused to improve the stability of the interaction with the target RNAsequence and to improve nuclease resistance.

In a non-limiting example, the introduction of chemically modifiednucleotides into nucleic acid molecules will provide a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example when compared toan all RNA nucleic acid molecule, the overall activity of the modifiednucleic acid molecule can be greater than the native molecule due toimproved stability and/or delivery of the molecule. Unlike nativeunmodified siRNA, chemically modified siRNA can also minimize thepossibility of activating interferon activity in humans.

In one embodiment, the invention features a chemically modified shortinterfering RNA (siRNA) molecule capable of mediating RNA interference(RNAi) against ADORA1 inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more nucleotidescomprising a backbone modified internucleotide linkage having Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally occurring or chemicallymodified, each X and Y is independently O, S, N, alkyl, or substitutedalkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl,O-alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W, X, Y and Z are notall O.

The chemically modified internucleotide linkages having Formula I, forexample wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siRNAduplex, for example in the sense strand, antisense strand, or bothstrands. The siRNA molecules of the invention can comprise one or morechemically modified internucleotide linkages having Formula I at the3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand, antisensestrand, or both strands. For example, an exemplary siRNA molecule of theinvention can comprise between about 1 and about 5 or more (e.g., about1, 2, 3, 4, 5, or more) chemically modified internucleotide linkageshaving Formula I at the 5′-end of the sense strand, antisense strand, orboth strands. In another non-limiting example, an exemplary siRNAmolecule of the invention can comprise one or more (e.g., about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemicallymodified internucleotide linkages having Formula I in the sense strand,antisense strand, or both strands. In yet another non-limiting example,an exemplary siRNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically modified internucleotide linkages having Formula I inthe sense strand, antisense strand, or both strands. In anotherembodiment, a siRNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically modified nucleotideor non-nucleotide having any of Formulae II, III, V, or VI.

In one embodiment, the invention features a chemically modified shortinterfering RNA (siRNA) molecule capable of mediating RNA interference(RNAi) against ADORA1 inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more nucleotides ornon-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-OH, alkyl-SH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula I; R9 is O,S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine,guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine,2,6-diaminopurine, or any other non-naturally occurring base that can becomplementary or non-complementary to ADORA1 RNA or a non-nucleosidicbase such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole,nebularine, pyridone, pyridinone, or any other non-naturally occurringuniversal base that can be complementary or non-complementary to ADORA1RNA.

The chemically modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siRNA duplex,for example in the sense strand, antisense strand, or both strands. ThesiRNA molecules of the invention can comprise one or more chemicallymodified nucleotide or non-nucleotide of Formula II at the 3 ‘-end,5’-end, or both 3′ and 5′-ends of the sense strand, antisense strand, orboth strands. For example, an exemplary siRNA molecule of the inventioncan comprise between about 1 and about 5 or more (e.g., about 1, 2, 3,4, 5, or more) chemically modified nucleotide or non-nucleotide ofFormula II at the 5 ‘-end of the sense strand, antisense strand, or bothstrands. In anther non-limiting example, an exemplary siRNA molecule ofthe invention can comprise between about 1 and about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically modified nucleotide ornon-nucleotide of Formula II at the 3’-end of the sense strand,antisense strand, or both strands.

In one embodiment, the invention features a chemically modified shortinterfering RNA (siRNA) molecule capable of mediating RNA interference(RNAi) against ADORA1 inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more nucleotides ornon-nucleotides having Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-OH, alkyl-SH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula I; R9 is O,S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine,guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine,2,6-diaminopurine, or any other non-naturally occurring base that can beemployed to be complementary or non-complementary to ADORA1 RNA or anon-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to ADORA1 RNA.

The chemically modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siRNA duplex,for example in the sense strand, antisense strand, or both strands. ThesiRNA molecules of the invention can comprise one or more chemicallymodified nucleotide or non-nucleotide of Formula III at the 3′-end,5′-end, or both 3′ and 5′-ends of the sense strand, antisense strand, orboth strands. For example, an exemplary siRNA molecule of the inventioncan comprise between about 1 and about 5 or more (e.g., about 1, 2, 3,4, 5, or more) chemically modified nucleotide or non-nucleotide ofFormula III at the 5′-end of the sense strand, antisense strand, or bothstrands. In anther non-limiting example, an exemplary siRNA molecule ofthe invention can comprise between about 1 and about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically modified nucleotide ornon-nucleotide of Formula III at the 3′-end of the sense strand,antisense strand, or both strands.

In another embodiment, a siRNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siRNA constructin a 3′,3′, 3′-2′, 2′-3′, or 5′,5′ configuration, such as at the 3′-end,5′-end, or both 3′ and 5′ ends of one or both siRNA strands.

In one embodiment, the invention features a chemically modified shortinterfering RNA (siRNA) molecule capable of mediating RNA interference(RNAi) against ADORA1 inside a cell or reconstituted in vitro system,wherein the chemical modification comprises a 5′-terminal phosphategroup having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo; each Z and W is independently O, S, N, alkyl, substitutedalkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or alkylhalo; and wherein W,X, Y and Z are not all O.

In one embodiment, the invention features a siRNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example a strand complementary toADORA1 RNA, wherein the siRNA molecule comprises an all RNA siRNAmolecule. In another embodiment, the invention features a siRNA moleculehaving a 5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siRNA molecule also comprises1-3 (e.g., 1, 2, or 3) nucleotide 3′-overhangs having between about 1and about 4 (e.g., about 1, 2, 3, or 4) deoxyribonucleotides on the3′-end of one or both strands. In another embodiment, a 5′-terminalphosphate group having Formula IV is present on the target-complementarystrand of a siRNA molecule of the invention, for example a siRNAmolecule having chemical modifications having Formula I, Formula IIand/or Formula III.

In one embodiment, the invention features a chemically modified shortinterfering RNA (siRNA) molecule capable of mediating RNA interference(RNAi) against ADORA1 inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more phosphorothioateinternucleotide linkages. For example, in a non-limiting example, theinvention features a chemically modified short interfering RNA (siRNA)having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioateinternucleotide linkages in one siRNA strand. In yet another embodiment,the invention features a chemically modified short interfering RNA(siRNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or morephosphorothioate internucleotide linkages in both siRNA strands. Thephosphorothioate internucleotide linkages can be present in one or botholigonucleotide strands of the siRNA duplex, for example in the sensestrand, antisense strand, or both strands. The siRNA molecules of theinvention can comprise one or more phosphorothioate internucleotidelinkages at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sensestrand, antisense strand, or both strands. For example, an exemplarysiRNA molecule of the invention can comprise between about 1 and about 5or more (e.g., about 1, 2, 3, 4, 5, or more) consecutivephosphorothioate internucleotide linkages at the 5′-end of the sensestrand, antisense strand, or both strands. In another non-limitingexample, an exemplary siRNA molecule of the invention can comprise oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidinephosphorothioate internucleotide linkages in the sense strand, antisensestrand, or both strands. In yet another non-limiting example, anexemplary siRNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purinephosphorothioate internucleotide linkages in the sense strand, antisensestrand, or both strands.

In one embodiment, the invention features a siRNA molecule, wherein thesense strand comprises one or more, for example about 1, 2, 3, 4, 5, 6,7, 8 , 9 , 10 or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5 ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3′, 5′, or both 3′ and 5′-ends of the sense strand; andwherein the antisense strand comprises any of between 1 and 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8 , 9 , 10 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the 3′,5′, or both 3′ and 5′-ends of the antisense strand. In anotherembodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more pyrimidine nucleotides of the sense and/or antisense siRNA standare chemically modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′, 5′,or both 3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siRNA molecule, whereinthe sense strand comprises between about 1 and about 5, specificallyabout 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3′, 5′, or both 3′ and 5′-ends of the sense strand; andwherein the antisense strand comprises any of between about 1 and about5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more(e.g., about 1, 2, 3, 4, 5 or more) universal base modified nucleotides,and optionally a terminal cap molecule at the 3′, 5′, or both 3′ and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidinenucleotides of the sense and/or antisense siRNA stand are chemicallymodified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without between about 1 and about 5 or more, forexample about 1, 2, 3, 4, 5 or more phosphorothioate internucleotidelinkages and/or a terminal cap molecule at the 3′, 5′, or both 3′ and5′-ends, being present in the same or different strand.

In one embodiment, the invention features a siRNA molecule, wherein thesense strand comprises one or more, for example about 1, 2, 3, 4, 5, 6,7, 8 , 9 , 10 or more phosphorothioate internucleotide linkages, and/orbetween one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy,2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 34, 5, or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′, 5′, or both 3′ and 5′-ends of the sensestrand; and wherein the antisense strand comprises any of between about1 and about 10, specifically about 1, 2, 3, 4, 5, 6, 7, 8 , 9 , 10 ormore phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3′, 5′, or both 3′ and 5′-ends of the antisense strand.In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisensesiRNA stand are chemically modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′, 5′,or both 3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siRNA molecule, whereinthe sense strand comprises between about 1 and about 5 or more,specifically about 1, 2, 3, 4, 5 or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more(e.g., about 1, 2, 3, 4, 5 or more) universal base modified nucleotides,and optionally a terminal cap molecule at the 3′, 5′, or both 3′ and5′-ends of the sense strand; and wherein the antisense strand comprisesany of between about 1 and about 5 or more, specifically about 1, 2, 3,4, 5 or more phosphorothioate internucleotide linkages, and/or one ormore (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3′, 5′, or both 3′ and 5′-ends of the antisense strand.In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisensesiRNA stand are chemically modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without between about 1 andabout 5, for example about 1, 2, 3, 4, 5 or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′, 5′,or both 3′ and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a chemically modified shortinterfering RNA (siRNA) molecule having between about 1 and about 5,specifically about 1, 2, 3, 4, 5 or more phosphorothioateinternucleotide linkages in each strand of the siRNA molecule.

In another embodiment, the invention features a siRNA moleculecomprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotidelinkage(s) can be at the 5′-end, 3′-end, or both 5′ and 3′ ends of oneor both siRNA sequence strands. In addition, the 2′-5′ internucleotidelinkage(s) can be present at various other positions within one or bothsiRNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more including every internucleotide linkage of a pyrimidinenucleotide in one or both strands of the siRNA molecule can comprise a2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a purine nucleotidein one or both strands of the siRNA molecule can comprise a 2′-5′internucleotide linkage.

In another embodiment, a chemically modified siRNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically modified, wherein each strand is between about 18 andabout 27 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27)nucleotides in length, wherein the duplex has between about 18 and about23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein thechemical modification comprises a structure having Formula I, FormulaII, Formula III and/or Formula IV. For example, an exemplary chemicallymodified siRNA molecule of the invention comprises a duplex having twostrands, one or both of which can be chemically modified with a chemicalmodification having Formula I, Formula II, Formula III, and/or FormulaIV, wherein each strand consists of 21 nucleotides, each having 2nucleotide 3′-overhangs, and wherein the duplex has 19 base pairs.

In another embodiment, a siRNA molecule of the invention comprises asingle stranded hairpin structure, wherein the siRNA is between about 36and about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotidesin length having between about 18 and about 23 (e.g., about 18, 19, 20,21, 22, or 23) base pairs, and wherein the siRNA can include a chemicalmodification comprising a structure having Formula I, Formula II,Formula III and/or Formula IV. For example, an exemplary chemicallymodified siRNA molecule of the invention comprises a linearoligonucleotide having between about 42 and about 50 (e.g., about 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemicallymodified with a chemical modification having Formula I, Formula II,Formula III, and/or Formula IV, wherein the linear oligonucleotide formsa hairpin structure having 19 base pairs and a 2 nucleotide 3′-overhang.

In another embodiment, a linear hairpin siRNA molecule of the inventioncontains a stem loop motif, wherein the loop portion of the siRNAmolecule is biodegradable. For example, a linear hairpin siRNA moleculeof the invention is designed such that degradation of the loop portionof the siRNA molecule in vivo can generate a double stranded siRNAmolecule with 3′-overhangs, such as 3′-overhangs comprising about 2nucleotides.

In another embodiment, a siRNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siRNA is between about 38and about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotidesin length having between about 18 and about 23 (e.g., about 18, 19, 20,21, 22, or 23) base pairs, and wherein the siRNA can include a chemicalmodification, which comprises a structure having Formula I, Formula II,Formula III and/or Formula IV. For example, an exemplary chemicallymodified siRNA molecule of the invention comprises a circularoligonucleotide having between about 42 and about 50 (e.g., about 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemicallymodified with a chemical modification having Formula I, Formula II,Formula III, and/or Formula IV, wherein the circular oligonucleotideforms a dumbbell shaped structure having 19 base pairs and 2 loops.

In another embodiment, a circular siRNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siRNAmolecule is biodegradable. For example, a circular siRNA molecule of theinvention is designed such that degradation of the loop portions of thesiRNA molecule in vivo can generate a double stranded siRNA moleculewith 3′-overhangs, such as 3′-overhangs comprising about 2 nucleotides.

In one embodiment, a siRNA molecule of the invention comprises at leastone abasic residue, for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula I; R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, a siRNA molecule of the invention comprises at leastone inverted abasic residue, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula I; R9 is O, S, CH2, S═O, CHF, or CF2, and either R2, R3, R8 orR13 serve as points of attachment to the siRNA molecule of theinvention.

In another embodiment, a siRNA molecule of the invention comprises anabasic residue having Formula II or III, wherein the abasic residuehaving Formula II or III is connected to the siRNA construct in a 3′,3′,3′-2′, 2′-3′, or 5′,5′ configuration, such as at the 3′-end, 5′-end, orboth 3′ and 5′ ends of one or both siRNA strands.

In one embodiment, a siRNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example at the 5′-end, 3′-end, 5′ and3′-end, or any combination thereof, of the siRNA molecule.

In another embodiment, a siRNA molecule of the invention comprises oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example at the 5′-end, 3′-end, 5′ and 3′-end, or anycombination thereof, of the siRNA molecule.

In one embodiment, the invention features a chemically modified shortinterfering RNA (siRNA) molecule capable of mediating RNA interference(RNAi) against ADORA1 inside a cell or reconstituted in vitro system,wherein the chemical modification comprises a conjugate covalentlyattached to the siRNA molecule. In another embodiment, the conjugate iscovalently attached to the siRNA molecule via a biodegradable linker. Inone embodiment, the conjugate molecule is attached at the 3′-end ofeither the sense strand, antisense strand, or both strands of the siRNA.In another embodiment, the conjugate molecule is attached at the 5′-endof either the sense strand, antisense strand, or both strands of thesiRNA. In yet another embodiment, the conjugate molecule is attachedboth the 3′-end and 5′-end of either the sense strand, antisense strand,or both strands of the siRNA, or any combination thereof. In oneembodiment, a conjugate molecule of the invention comprises a moleculethat facilitates delivery of a siRNA molecule into a biological systemsuch as a cell. In another embodiment, the conjugate molecule attachedto the siRNA is a poly ethylene glycol, human serum albumin, or a ligandfor a cellular receptor that can mediate cellular uptake. Examples ofspecific conjugate molecules contemplated by the instant invention thatcan be attached to siRNA molecules are described in Vargeese et al.,U.S. Ser. No. 60/311,865, incorporated by reference herein.

In one embodiment, the invention features a siRNA molecule capable ofmediating RNA interference (RNAi) against ADORA1 inside a cell orreconstituted in vitro system, wherein one or both strands of the siRNAcomprise ribonucleotides at positions withing the siRNA that arecritical for siRNA mediated RNAi in a cell. All other positions withinthe siRNA can include chemically modified nucleotides and/ornon-nucleotides such as nucleotides and or non-nucleotides havingFormula I, II, III, IV, V, or VI, or any combination thereof to theextent that the ability of the siRNA molecule to support RNAi activityin a cell is maintained.

In one embodiment, the invention features a method for modulating theexpression of an ADORA1 gene within a cell, comprising: (a) synthesizinga siRNA molecule of the invention, which can be chemically modified,wherein one of the siRNA strands includes a sequence complementary toRNA of the ADORA1 gene; and (b) introducing the siRNA molecule into acell under conditions suitable to modulate the expression of the ADORA1gene in the cell.

In one embodiment, the invention features a method for modulating theexpression of an ADORA1 gene within a cell, comprising: (a) synthesizinga siRNA molecule of the invention, which can be chemically modified,wherein one of the siRNA strands includes a sequence complementary toRNA of the ADORA1 gene and wherein the sense strand sequence of thesiRNA is identical to the complementary sequence of the ADORA1 RNA; and(b) introducing the siRNA molecule into a cell under conditions suitableto modulate the expression of the ADORA1 gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one ADORA1 gene within a cell, comprising:(a) synthesizing siRNA molecules of the invention, which can bechemically modified, wherein one of the siRNA strands includes asequence complementary to RNA of the ADORA1 genes; and (b) introducingthe siRNA molecules into a cell under conditions suitable to modulatethe expression of the ADORA1 genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one ADORA1 gene within a cell, comprising:(a) synthesizing a siRNA molecule of the invention, which can bechemically modified, wherein one of the siRNA strands includes asequence complementary to RNA of the ADORA1 gene and wherein the sensestrand sequence of the siRNA is identical to the complementary sequenceof the ADORA1 RNA; and (b) introducing the siRNA molecules into a cellunder conditions suitable to modulate the expression of the ADORA1 genesin the cell.

In one embodiment, the invention features a method of modulating theexpression of an ADORA1 gene in a tissue explant, comprising: (a)synthesizing a siRNA molecule of the invention, which can be chemicallymodified, wherein one of the siRNA strands includes a sequencecomplementary to RNA of the ADORA1 gene; (b) introducing the siRNAmolecule into a cell of the tissue explant derived from a particularorganism under conditions suitable to modulate the expression of theADORA1 gene in the tissue explant, and (c) optionally introducing thetissue explant back into the organism the tissue was derived from orinto another organism under conditions suitable to modulate theexpression of the ADORA1 gene in that organism.

In one embodiment, the invention features a method of modulating theexpression of an ADORA1 gene in a tissue explant, comprising: (a)synthesizing a siRNA molecule of the invention, which can be chemicallymodified, wherein one of the siRNA strands includes a sequencecomplementary to RNA of the ADORA1 gene and wherein the sense strandsequence of the siRNA is identical to the complementary sequence of theADORA1 RNA; (b) introducing the siRNA molecule into a cell of the tissueexplant derived from a particular organism under conditions suitable tomodulate the expression of the ADORA1 gene in the tissue explant, and(c) optionally introducing the tissue explant back into the organism thetissue was derived from or into another organism under conditionssuitable to modulate the expression of the ADORA1 gene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one ADORA1 gene in a tissue explant, comprising:(a) synthesizing siRNA molecules of the invention, which can bechemically modified, wherein one of the siRNA strands includes asequence complementary to RNA of the ADORA1 genes; (b) introducing thesiRNA molecules into a cell of the tissue explant derived from aparticular organism under conditions suitable to modulate the expressionof the ADORA1 genes in the tissue explant, and (c) optionallyintroducing the tissue explant back into the organism the tissue wasderived from or into another organism under conditions suitable tomodulate the expression of the ADORA1 genes in that organism.

In one embodiment, the invention features a method of modulating theexpression of an ADORA1 gene in an organism, comprising: (a)synthesizing a siRNA molecule of the invention, which can be chemicallymodified, wherein one of the siRNA strands includes a sequencecomplementary to RNA of the ADORA1 gene; and (b) introducing the siRNAmolecule into the organism under conditions suitable to modulate theexpression of the ADORA1 gene in the organism.

In another embodiment, the invention features a method of modulating theexpression of more than one ADORA1 gene in an organism, comprising: (a)synthesizing siRNA molecules of the invention, which can be chemicallymodified, wherein one of the siRNA strands includes a sequencecomplementary to RNA of the ADORA1 genes; and (b) introducing the siRNAmolecules into the organism under conditions suitable to modulate theexpression of the ADORA1 genes in the organism.

The siRNA molecules of the invention can be designed to inhibit ADORA1gene expression through RNAi targeting of a variety of RNA molecules. Inone embodiment, the siRNA molecules of the invention are used to targetvarious RNAs corresponding to a target gene. Non-limiting examples ofsuch RNAs include messenger RNA (mRNA), alternate RNA splice variants oftarget gene(s), post-transcriptionally modified RNA of target gene(s),pre-mRNA of target gene(s), and/or RNA templates used for ADORA1activity. If alternate splicing produces a family of transcripts thatare distinguished by usage of appropriate exons, the instant inventioncan be used to inhibit gene expression through the appropriate exons tospecifically inhibit or to distinguish among the functions of genefamily members. For example, a protein that contains an alternativelyspliced transmembrane domain can be expressed in both membrane bound andsecreted forms. Use of the invention to target the exon containing thetransmembrane domain can be used to determine the functionalconsequences of pharmaceutical targeting of membrane bound as opposed tothe secreted form of the protein. Non-limiting examples of applicationsof the invention relating to targeting these RNA molecules includetherapeutic pharmaceutical applications, pharmaceutical discoveryapplications, molecular diagnostic and gene function applications, andgene mapping, for example using single nucleotide polymorphism mappingwith siRNA molecules of the invention. Such applications can beimplemented using known gene sequences or from partial sequencesavailable from an expressed sequence tag (EST).

In another embodiment, the siRNA molecules of the invention are used totarget conserved sequences corresponding to a gene family or genefamilies such as checkpoint kinase genes. As such, siRNA moleculestargeting multiple checkpoint kinase targets can provide increasedtherapeutic effect. In addition, siRNA can be used to characterizepathways of gene function in a variety of applications. For example, thepresent invention can be used to inhibit the activity of target gene(s)in a pathway to determine the function of uncharacterized gene(s) ingene function analysis, mRNA function analysis, or translationalanalysis. The invention can be used to determine potential target genepathways involved in various diseases and conditions towardpharmaceutical development. The invention can be used to understandpathways of gene expression involved in development, such as prenataldevelopment, postnatal development and/or aging.

In one embodiment, siRNA molecule(s) and/or methods of the invention areused to inhibit the expression of gene(s) that encode RNA referred to byGenbank Accession number, for example genes such as Genbank AccessionNo. NM_(—)000674. Such sequences are readily obtained using this GenbankAccession number.

In one embodiment, the invention features a method comprising: (a)generating a randomized library of siRNA constructs having apredetermined complexity, such as of 4^(N), where N represents thenumber of base paired nucleotides in each of the siRNA construct strands(eg. for a siRNA construct having 21 nucleotide sense and antisensestrands with 19 base pairs, the complexity would be 4¹⁹); and (b)assaying the siRNA constructs of (a) above, under conditions suitable todetermine RNAi target sites within the target ADORA1 RNA sequence. Inanother embodiment, the siRNA molecules of (a) have strands of a fixedlength, for example about 23 nucleotides in length. In yet anotherembodiment, the siRNA molecules of (a) are of differing length, forexample having strands of about 19 to about 25 (e.g., about 19, 20, 21,22, 23, 24, or 25) nucleotides in length. In yet another embodiment, theassay can comprise a reconstituted in vitro siRNA assay as described inExample 6 herein. In another embodiment, the assay can comprise a cellculture system in which target RNA is expressed. In another embodiment,fragments of ADORA1 RNA are analyzed for detectable levels of cleavage,for example by gel electrophoresis, northern blot analysis, or RNAseprotection assays, to determine the most suitable target site(s) withinthe target ADORA1 RNA sequence. In another embodiment, the target ADORA1RNA sequence can be obtained as is known in the art, for example, bycloning and/or transcription for in vitro systems, and by cellularexpression in in vivo systems.

In another embodiment, the invention features a method comprising: (a)analyzing the sequence of a RNA target encoded by an ADORA1 gene; (b)synthesizing one or more sets of siRNA molecules having sequencecomplementary to one or more regions of the RNA of (a); and (c) assayingthe siRNA molecules of (b) under conditions suitable to determine RNAitargets within the target RNA sequence. In another embodiment, the siRNAmolecules of (b) have strands of a fixed length, for example about 23nucleotides in length. In yet another embodiment, the siRNA molecules of(b) are of differing length, for example having strands of about 19 toabout 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides inlength. In yet another embodiment, the assay can comprise areconstituted in vitro siRNA assay as described in Example 6 herein. Inanother embodiment, the assay can comprise a cell culture system inwhich target RNA is expressed. Fragments of ADORA1 RNA are analyzed fordetectable levels of cleavage, for example by gel electrophoresis,northern blot analysis, or RNAse protection assays, to determine themost suitable target site(s) within the target ADORA1 RNA sequence. Thetarget ADORA1 RNA sequence can be obtained as is known in the art, forexample, by cloning and/or transcription for in vitro systems, and byexpression in in vivo systems.

By “target site” is meant a sequence within a target RNA that is“targeted” for cleavage mediated by a siRNA construct which containssequences within its antisense region that are complementary to thetarget sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising asiRNA molecule of the invention, which can be chemically modified, in apharmaceutically acceptable carrier or diluent. In another embodiment,the invention features a pharmaceutical composition comprising siRNAmolecules of the invention, which can be chemically modified, targetingone or more genes in a pharmaceutically acceptable carrier or diluent.In another embodiment, the invention features a method for treating orpreventing a disease or condition in a subject, comprising administeringto the subject a composition of the invention under conditions suitablefor the treatment or prevention of the disease or condition in thesubject, alone or in conjunction with one or more other therapeuticcompounds.

In another embodiment, the invention features a method for validating anADORA1 gene target, comprising: (a) synthesizing a siRNA molecule of theinvention, which can be chemically modified, wherein one of the siRNAstrands includes a sequence complementary to RNA of an ADORA1 targetgene; (b) introducing the siRNA molecule into a cell, tissue, ororganism under conditions suitable for modulating expression of theADORA1 target gene in the cell, tissue, or organism; and (c) determiningthe function of the gene by assaying for any phenotypic change in thecell, tissue, or organism.

In one embodiment, the invention features a kit containing a siRNAmolecule of the invention, which can be chemically modified, that can beused to modulate the expression of an ADORA1 target gene in a cell,tissue, or organism. In another embodiment, the invention features a kitcontaining more than one siRNA molecule of the invention, which can bechemically modified, that can be used to modulate the expression of morethan one ADORA1 target gene in a cell, tissue, or organism.

In one embodiment, the invention features a cell containing one or moresiRNA molecules of the invention, which can be chemically modified. Inanother embodiment, the cell containing a siRNA molecule of theinvention is a mammalian cell. In yet another embodiment, the cellcontaining a siRNA molecule of the invention is a human cell.

In one embodiment, the synthesis of a siRNA molecule of the invention,which can be chemically modified, comprises: (a) synthesis of twocomplementary strands of the siRNA molecule; (b) annealing the twocomplementary strands together under conditions suitable to obtain adouble stranded siRNA molecule. In another embodiment, synthesis of thetwo complementary strands of the siRNA molecule is by solid phaseoligonucleotide synthesis. In yet another embodiment, synthesis of thetwo complementary strands of the siRNA molecule is by solid phase tandemoligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing asiRNA duplex molecule comprising: (a) synthesizing a firstoligonucleotide sequence strand of the siRNA molecule, wherein the firstoligonucleotide sequence strand comprises a cleavable linker moleculethat can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siRNA; (b) synthesizing thesecond oligonucleotide sequence strand of siRNA on the scaffold of thefirst oligonucleotide sequence strand, wherein the secondoligonucleotide sequence strand further comprises a chemical moiety thancan be used to purify the siRNA duplex; (c) cleaving the linker moleculeof (a) under conditions suitable for the two siRNA oligonucleotidestrands to hybridize and form a stable duplex; and (d) purifying thesiRNA duplex utilizing the chemical moiety of the second oligonucleotidesequence strand. In another embodiment, cleavage of the linker moleculein (c) above takes place during deprotection of the oligonucleotide, forexample under hydrolysis conditions using an alkylamine base such asmethylamine. In another embodiment, the method of synthesis comprisessolid phase synthesis on a solid support such as controlled pore glass(CPG) or polystyrene, wherein the first sequence of (a) is synthesizedon a cleavable linker, such as a succinyl linker, using the solidsupport as a scaffold. The cleavable linker in (a) used as a scaffoldfor synthesizing the second strand can comprise similar reactivity asthe solid support derivatized linker, such that cleavage of the solidsupport derivatized linker and the cleavable linker of (a) takes placeconcomitantly. In another embodiment, the chemical moiety of (b) thatcan used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group, which can be employedin a trityl-on synthesis strategy as described herein. In yet anotherembodiment, the chemical moiety, such as a dimethoxytrityl group, isremoved during purification, for example using acidic conditions.

In a further embodiment, the method for siRNA synthesis is a solutionphase synthesis or hybrid phase synthesis wherein both strands of thesiRNA duplex are synthesized in tandem using a cleavable linker attachedto the first sequence which acts a scaffold for synthesis of the secondsequence. Cleavage of the linker under conditions suitable forhybridization of the separate siRNA sequence strands results information of the double stranded siRNA molecule.

In another embodiment, the invention features a method for synthesizinga siRNA duplex molecule comprising: (a) synthesizing one oligonucleotidesequence strand of the siRNA molecule, wherein the sequence comprises acleavable linker molecule that can be used as a scaffold for thesynthesis of another oligonucleotide sequence; (b) synthesizing a secondoligonucleotide sequence having complementarity to the first sequencestrand on the scaffold of (a), wherein the second sequence comprises theother strand of the double stranded siRNA molecule and wherein thesecond sequence further comprises a chemical moiety than can be used toisolate the attached oligonucleotide sequence; (c) purifying the productof (b) utilizing the chemical moiety of the second oligonucleotidesequence strand under conditions suitable for isolating the full lengthsequence comprising both siRNA oligonucleotide strands connected by thecleavable linker; and (d) under conditions suitable for the two siRNAoligonucleotide strands to hybridize and form a stable duplex. Inanother embodiment, cleavage of the linker molecule in (c) above takesplace during deprotection of the oligonucleotide, for example underhydrolysis conditions. In another embodiment, cleavage of the linkermolecule in (c) above takes place after deprotection of theoligonucleotide. In another embodiment, the method of synthesiscomprises solid phase synthesis on a solid support such as controlledpore glass (CPG) or polystyrene, wherein the first sequence of (a) issynthesized on a cleavable linker, such as a succinyl linker, using thesolid support as a scaffold. The cleavable linker in (a) used as ascaffold for synthesizing the second strand can comprise similarreactivity or differing reactivity as the solid support derivatizedlinker, such that cleavage of the solid support derivatized linker andthe cleavable linker of (a) takes place either concomitantly orsequentially. In another embodiment, the chemical moiety of (b) that canused to isolate the attached oligonucleotide sequence comprises a tritylgroup, for example a dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble stranded siRNA molecule in a single synthetic process,comprising: (a) synthesizing an oligonucleotide having a first and asecond sequence, wherein the first sequence is complementary to thesecond sequence, and the first oligonucleotide sequence is linked to thesecond sequence via a cleavable linker, and wherein a terminal5′-protecting group, for example a 5′-O-dimethoxytrityl group (5′-O-DMT)remains on the oligonucleotide having the second sequence; (b)deprotecting the oligonucleotide whereby the deprotection results in thecleavage of the linker joining the two oligonucleotide sequences; and(c) purifying the product of (b) under conditions suitable for isolatingthe double stranded siRNA molecule, for example using a trityl-onsynthesis strategy as described herein.

In one embodiment, the invention features siRNA constructs that mediateRNAi against ADORA1, wherein the siRNA construct comprises one or morechemical modifications, for example one or more chemical modificationshaving Formula I, II, III, IV, or V, that increases the nucleaseresistance of the siRNA construct.

In another embodiment, the invention features a method for generatingsiRNA molecules with increased nuclease resistance comprising (a)introducing nucleotides having any of Formula I-VI into a siRNAmolecule, and (b) assaying the siRNA molecule of step (a) underconditions suitable for isolating siRNA molecules having increasednuclease resistance.

In one embodiment, the invention features siRNA constructs that mediateRNAi against ADORA1, wherein the siRNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the sense and antisense strands of the siRNA construct.

In another embodiment, the invention features a method for generatingsiRNA molecules with increased binding affinity between the sense andantisense strands of the siRNA molecule comprising (a) introducingnucleotides having any of Formula I-VI into a siRNA molecule, and (b)assaying the siRNA molecule of step (a) under conditions suitable forisolating siRNA molecules having increased binding affinity between thesense and antisense strands of the siRNA molecule.

In one embodiment, the invention features siRNA constructs that mediateRNAi against ADORA1, wherein the siRNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siRNA construct and acomplementary target RNA sequence within a cell.

In another embodiment, the invention features a method for generatingsiRNA molecules with increased binding affinity between the antisensestrand of the siRNA molecule and a complementary target RNA sequence,comprising (a) introducing nucleotides having any of Formula I-VI into asiRNA molecule, and (b) assaying the siRNA molecule of step (a) underconditions suitable for isolating siRNA molecules having increasedbinding affinity between the antisense strand of the siRNA molecule anda complementary target RNA sequence.

In one embodiment, the invention features siRNA constructs that mediateRNAi against ADORA1, wherein the siRNA construct comprises one or morechemical modifications described herein that modulate the polymeraseactivity of a cellular polymerase capable of generating additionalendogenous siRNA molecules having sequence homology to the chemicallymodified siRNA construct.

In another embodiment, the invention features a method for generatingsiRNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siRNAmolecules having sequence homology to the chemically modified siRNAmolecule comprising (a) introducing nucleotides having any of FormulaI-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step(a) under conditions suitable for isolating siRNA molecules capable ofmediating increased polymerase activity of a cellular polymerase capableof generating additional endogenous siRNA molecules having sequencehomology to the chemically modified siRNA molecule.

In one embodiment, the invention features chemically modified siRNAconstructs that mediate RNAi against ADORA1 in a cell, wherein thechemical modifications do not significantly effect the interaction ofsiRNA with a target RNA molecule and/or proteins or other factors thatare essential for RNAi in a manner that would decrease the efficacy ofRNAi mediated by such siRNA constructs.

In another embodiment, the invention features a method for generatingsiRNA molecules with improved RNAi activity against ADORA1, comprising(a) introducing nucleotides having any of Formula I-VI into a siRNAmolecule, and (b) assaying the siRNA molecule of step (a) underconditions suitable for isolating siRNA molecules having improved RNAiactivity.

In yet another embodiment, the invention features a method forgenerating siRNA molecules with improved RNAi activity against an ADORA1target RNA, comprising (a) introducing nucleotides having any of FormulaI-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step(a) under conditions suitable for isolating siRNA molecules havingimproved RNAi activity against the target RNA.

In one embodiment, the invention features siRNA constructs that mediateRNAi against ADORA1, wherein the siRNA construct comprises one or morechemical modifications described herein that modulates the cellularuptake of the siRNA construct.

In another embodiment, the invention features a method for generatingsiRNA molecules against ADORA1 with improved cellular uptake, comprising(a) introducing nucleotides having any of Formula I-VI into a siRNAmolecule, and (b) assaying the siRNA molecule of step (a) underconditions suitable for isolating siRNA molecules having improvedcellular uptake.

In one embodiment, the invention features siRNA constructs that mediateRNAi against ADORA1, wherein the siRNA construct comprises one or morechemical modifications described herein that increases thebioavailability of the siRNA construct, for example by attachingpolymeric conjugates such as polyethyleneglycol or equivalent conjugatesthat improve the pharmacokinetics of the siRNA construct, or byattaching conjugates that target specific tissue types or cell types invivo. Non-limiting examples of such conjugates are described in Vargeeseet al., U.S. Ser. No. 60/311,865 incorporated by reference herein.

In one embodiment, the invention features a method for generating siRNAmolecules of the invention with improved bioavailability, comprising (a)introducing a conjugate into the structure of a siRNA molecule, and (b)assaying the siRNA molecule of step (a) under conditions suitable forisolating siRNA molecules having improved bioavailability. Suchconjugates can include ligands for cellular receptors such as peptidesderived from naturally occurring protein ligands, protein localizationsequences including cellular ZIP code sequences, antibodies, nucleicacid aptamers, vitamins and other co-factors such as folate andN-acetylgalactosamine, polymers such as polyethyleneglycol (PEG),phospholipids, polyamines such as spermine or spermidine, and others.

In another embodiment, the invention features a method for generatingsiRNA molecules of the invention with improved bioavailability,comprising (a) introducing an excipient formulation to a siRNA molecule,and (b) assaying the siRNA molecule of step (a) under conditionssuitable for isolating siRNA molecules having improved bioavailability.Such excipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, and others.

In another embodiment, the invention features a method for generatingsiRNA molecules of the invention with improved bioavailability,comprising (a) introducing nucleotides having any of Formula I-VI into asiRNA molecule, and (b) assaying the siRNA molecule of step (a) underconditions suitable for isolating siRNA molecules having improvedbioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to siRNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 2,000 to about 50,000daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include the siRNA and a vehiclethat promotes introduction of the siRNA. Such a kit can also includeinstructions to allow a user of the kit to practice the invention.

The term “short interfering RNA” or “siRNA” as used herein refers to anynucleic acid molecule capable of mediating RNA interference “RNAi” orgene silencing; see for example Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914. Non limiting examples of siRNA molecules ofthe invention are shown in FIG. 2. For example the siRNA can be a doublestranded polynucleotide molecule comprising self complementary sense andantisense regions, wherein the antisense region comprisescomplementarity to a target nucleic acid molecule. The siRNA can be asingle stranded hairpin polynucleotide having self complementary senseand antisense regions, wherein the antisense region comprisescomplementarity to a target nucleic acid molecule. The siRNA can be acircular single stranded polynucleotide having two or more loopstructures and a stem comprising self complementary sense and antisenseregions, wherein the antisense region comprises complementarity to atarget nucleic acid molecule, and wherein the circular polynucleotidecan be processed either in vivo or in vitro to generate an active siRNAcapable of mediating RNAi. As used herein, siRNA molecules need not belimited to those molecules containing only RNA, but further encompasseschemically modified nucleotides and non-nucleotides.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit” it is meant that the activity of a gene expression productor level of RNAs or equivalent RNAs encoding one or more gene productsis reduced below that observed in the absence of the nucleic acidmolecule of the invention. In one embodiment, inhibition with a siRNAmolecule preferably is below that level observed in the presence of aninactive or attenuated molecule that is unable to mediate an RNAiresponse. In another embodiment, inhibition of gene expression with thesiRNA molecule of the instant invention is greater in the presence ofthe siRNA molecule than in its absence.

By “gene” or “target gene” is meant, a nucleic acid that encodes an RNA,for example, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide. The target gene can be a genederived from a cell, an endogenous gene, a transgene, or exogenous genessuch as genes of a pathogen, for example a virus, which is present inthe cell after infection thereof. The cell containing the target genecan be derived from or contained in any organism, for example a plant,animal, protozoan, virus, bacterium, or fungus. Non-limiting examples ofplants include monocots, dicots, or gymnosperms. Non-limiting examplesof animals include vertebrates or invertebrates. Non-limiting examplesof fungi include molds or yeasts.

By “ADORA1” is meant, a polypeptide comprising an adenosine A1 receptoror polynucleotide encoding an Ets adenosine A1 receptor, for example apolynucleotide having Genbank Accession No. NM_(—)000674.

By “highly conserved sequence region” is meant, a nucleotide sequence ofone or more regions in a target gene does not vary significantly fromone generation to the other or from one biological system to the other.

By “complementarity” or “complementary” is meant that a nucleic acid canform hydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types of interaction.In reference to the nucleic molecules of the present invention, thebinding free energy for a nucleic acid molecule with its complementarysequence is sufficient to allow the relevant function of the nucleicacid to proceed, e.g., RNAi activity. For example, the degree ofcomplementarity between the sense and antisense strand of the siRNAconstruct can be the same or different from the degree ofcomplementarity between the antisense strand of the siRNA and the targetRNA sequence. Complementarity to the target sequence of less than 100%in the antisense strand of the siRNA duplex, including point mutations,is reported not to be tolerated when these changes are located betweenthe 3′-end and the middle of the antisense siRNA (completely abolishessiRNA activity), whereas mutations near the 5′-end of the antisensesiRNA strand can exhibit a small degree of RNAi activity (Elbashir etal., 2001, The EMBO Journal, 20, 6877-6888). Determination of bindingfree energies for nucleic acid molecules is well known in the art (see,e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frieret al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al.,1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarityindicates the percentage of contiguous residues in a nucleic acidmolecule that can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence.

The siRNA molecules of the invention represent a novel therapeuticapproach to treat a variety of allergic/inflammatory diseases andconditions, including but not limited to asthma, allergic rhinitis,atopic dermatitis, and other indications that can respond to the levelof ADORA1.

In one embodiment of the present invention, each sequence of a siRNAmolecule of the invention is independently about 18 to about 24nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22,23, or 24 nucleotides in length. In another embodiment, the siRNAduplexes of the invention independently comprise between about 17 andabout 23 (e.g., about 17, 18, 19, 20, 21, 22, or 23) base pairs. In yetanother embodiment, siRNA molecules of the invention comprising hairpinor circular structures are about 35 to about 55 (e.g., about 35, 40, 45,50, or 55) nucleotides in length, or about 38 to about 44 (e.g., about38, 39, 40, 41, 42, 43, or 44) nucleotides in length and comprisingabout 16 to about 22 (e.g., about 16, 17, 18, 19, 20, 21, or 22) basepairs. Exemplary siRNA molecules of the invention are shown in Table Iand III (all sequences are shown 5′-3′) and/or FIGS. 4 and 5.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, andcats. The cell can be eukaryotic (e.g., a mammalian cell, such as ahuman cell). The cell can be of somatic or germ line origin, totipotentor pluripotent, dividing or non-dividing. The cell can also be derivedfrom or can comprise a gamete or embryo, a stem cell, or a fullydifferentiated cell.

The siRNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through injection, infusion pump or stent, with or without theirincorporation in biopolymers. In particular embodiments, the nucleicacid molecules of the invention comprise sequences shown in Table I, IIIand/or FIGS. 4 and 5. Examples of such nucleic acid molecules consistessentially of sequences defined in these tables/figures.

In another aspect, the invention provides mammalian cells containing oneor more siRNA molecules of this invention. The one or more siRNAmolecules can independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribo-furanose moiety. The terms includedouble stranded RNA, single stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siRNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. In one embodiment, a subject is a mammal or mammaliancells. In another embodiment, a subject is a human or human cells.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar, for example where any of the ribosecarbons (C1, C2, C3, C4, or C5), are independently or in combinationabsent from the nucleotide.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to treatdiseases or conditions discussed herein. For example, to treat aparticular disease or condition, the siRNA molecules can be administeredto a subject or can be administered to other appropriate cells evidentto those skilled in the art, individually or in combination with one ormore drugs under conditions suitable for the treatment.

In a further embodiment, the siRNA molecules can be used in combinationwith other known treatments to treat conditions or diseases discussedabove. For example, the described molecules could be used in combinationwith one or more known therapeutic agents to treat a disease orcondition. Non-limiting examples of other therapeutic agents that can bereadily combined with a siRNA molecule of the invention are enzymaticnucleic acid molecules, allosteric nucleic acid molecules, antisense,decoy, or aptamer nucleic acid molecules, antibodies such as monoclonalantibodies, small molecules, and other organic and/or inorganiccompounds including metals, salts and ions.

In one embodiment, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one siRNA moleculeof the invention, in a manner which allows expression of the siRNAmolecule. For example, the vector can contain sequence(s) encoding bothstrands of a siRNA molecule comprising a duplex. The vector can alsocontain sequence(s) encoding a single nucleic acid molecule that is selfcomplementary and thus forms a siRNA molecule. Non-limiting examples ofsuch expression vectors are described in Paul et al., 2002, NatureBiotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology,19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina etal., 2002, Nature Medicine, advance online publicationdoi:10.1038/nm725.

In another embodiment, the invention features a mammalian cell, forexample, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the inventioncomprises a sequence for a siRNA molecule having complementarity to aRNA molecule referred to by a Genbank Accession numbers, for examplegenes such as Genbank Accession No. No. NM_(—)000674.

In one embodiment, an expression vector of the invention comprises anucleic acid sequence encoding two or more siRNA molecules, which can bethe same or different.

In another aspect of the invention, siRNA molecules that interact withtarget RNA molecules and down-regulate gene encoding target RNAmolecules (for example target RNA molecules referred to by GenbankAccession numbers herein) are expressed from transcription unitsinserted into DNA or RNA vectors. The recombinant vectors can be DNAplasmids or viral vectors. siRNA expressing viral vectors can beconstructed based on, but not limited to, adeno-associated virus,retrovirus, adenovirus, or alphavirus. The recombinant vectors capableof expressing the siRNA molecules can be delivered as described herein,and persist in target cells. Alternatively, viral vectors can be usedthat provide for transient expression of siRNA molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siRNAmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of siRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from a subject followed byreintroduction into the subject, or by any other means that would allowfor introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

By “comprising” is meant including, but not limited to, whatever followsthe word “comprising”. Thus, use of the term “comprising” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and may or may not be present. By “consisting of” is meantincluding, and limited to, whatever follows the phrase “consisting of”.Thus, the phrase “consisting of” indicates that the listed elements arerequired or mandatory, and that no other elements may be present. By“consisting essentially of” is meant including any elements listed afterthe phrase, and limited to other elements that do not interfere with orcontribute to the activity or action specified in the disclosure for thelisted elements. Thus, the phrase “consisting essentially of” indicatesthat the listed elements are required or mandatory, but that otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

Other features and advantages of the invention will be apparent from thefollowing description of the exemplary embodiments thereof, and from theclaims.

First the drawings will be described briefly.

DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis ofsiRNA molecules. The complementary siRNA sequence strands, strand 1 andstrand 2, are synthesized in tandem and are connected by a cleavablelinkage, such as a nucleotide succinate or abasic succinate, which canbe the same or different from the cleavable linker used for solid phasesynthesis on a solid support. The synthesis can be either solid phase orsolution phase, in the example shown, the synthesis is a solid phasesynthesis. The synthesis is performed such that a protecting group, suchas a dimethoxytrityl group, remains intact on the terminal nucleotide ofthe tandem oligonucleotide. Upon cleavage and deprotection of theoligonucleotide, the two siRNA strands spontaneously hybridize to form asiRNA duplex, which allows the purification of the duplex by utilizingthe properties of the terminal protecting group, for example by applyinga trityl on purification method wherein only duplexes/oligonucleotideswith the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOV mass spectrum of a purified siRNA duplexsynthesized by a method of the invention. The two peaks shown correspondto the predicted mass of the separate siRNA sequence strands. Thisresult demonstrates that the siRNA duplex generated from tandemsynthesis can be purified as a single entity using a simple trityl-onpurification methodology.

FIG. 3 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double stranded RNA (dsRNA),which is generated by RNA dependent RNA polymerase (RdRP) from foreignsingle stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme which in turn generates siRNA duplexeshaving terminal phosphate groups (P). An active siRNA complex formswhich recognizes a target RNA, resulting in degradation of the targetRNA by the RISC endonuclease complex or in the synthesis of additionalRNA by RNA dependent RNA polymerase (RdRP), which can activate DICER andresult in additional siRNA molecules, thereby amplifying the RNAiresponse.

FIG. 4 shows non-limiting examples of chemically modified siRNAconstructs of the present invention. In the figure, N stands for anynucleotide (adenosine, guanosine, cytosine, uridine, or optionallythymidine, for example thymidine can be substituted in the overhangingregions designated by parenthesis (N N). Various modifications are shownfor the sense and antisense strands of the siRNA constructs. A The sensestrand comprises 21 nucleotides having four phosphorothioate 5′ and3′-terminal internucleotide linkages, wherein the two terminal3′-nucleotides are optionally base paired and wherein all pyrimidinenucleotides that may be present are 2′-O-methyl modified nucleotidesexcept for (N N) nucleotides, which can comprise naturally occurringribonucleotides, deoxynucleotides, universal bases, or other chemicalmodifications described herein. The antisense strand comprises 21nucleotides, wherein the two terminal 3′-nucleotides are optionallycomplimentary to the target RNA sequence, and having one 3′-terminalphosphorothioate internucleotide linkage and four 5′-terminalphosphorothioate internucleotide linkages and wherein all pyrimidinenucleotides that may be present are 2′-deoxy-2′-fluoro modifiednucleotides except for (N N) nucleotides, which can comprise naturallyoccurring ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. B The sense strand comprises 21nucleotides wherein the two terminal 3′-nucleotides are optionally basepaired and wherein all pyrimidine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise naturally occurring ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, wherein the two terminal3′-nucleotides are optionally complimentary to the target RNA sequence,and wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides,which can comprise naturally occurring ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. C The sense strand comprises 21 nucleotides having 5′-and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise naturally occurring ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. The antisense strand comprises 21 nucleotides, whereinthe two terminal 3′-nucleotides are optionally complimentary to thetarget RNA sequence, and wherein all pyrimidine nucleotides that may bepresent are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise naturally occurring ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. D The sense strand comprises 21 nucleotides havingfive phosphorothioate 5′ and 3′-terminal internucleotide linkages,wherein the two terminal 3′-nucleotides are optionally base paired andwherein all nucleotides are ribonucleotides except for (N N)nucleotides, which can comprise naturally occurring ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. The antisense strand comprises 21 nucleotides, whereinthe two terminal 3′-nucleotides are optionally complimentary to thetarget RNA sequence, and having one 3′-terminal phosphorothioateinternucleotide linkage and five 5′-terminal phosphorothioateinternucleotide linkages and wherein all nucleotides are ribonucleotidesexcept for (N N) nucleotides, which can comprise naturally occurringribonucleotides, deoxynucleotides, universal bases, or other chemicalmodifications described herein. E The sense strand comprises 21nucleotides wherein the two terminal 3′-nucleotides are optionally basepaired and wherein all pyrimidine nucleotides that may be present are2′-O-methyl nucleotides except for (N N) nucleotides, which can comprisenaturally occurring ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. The antisense strandcomprises 21 nucleotides all having phosphorothioate internucleotidelinkages, wherein the two terminal 3′-nucleotides are optionallycomplimentary to the target RNA sequence, and wherein all nucleotidesare ribonucleotides except for (N N) nucleotides, which can comprisenaturally occurring ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. F The sense strandcomprises 21 nucleotides having 5′- and 3′-terminal cap moieties,wherein the two terminal 3′-nucleotides are optionally base paired andwherein all pyrimidine nucleotides that may be present are 2′-O-methylnucleotides except for (N N) nucleotides, which can comprise naturallyoccurring ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. The antisense strand comprises21 nucleotides, wherein the two terminal 3′-nucleotides are optionallycomplimentary to the target RNA sequence, and having one 3′-terminalphosphorothioate internucleotide linkage and wherein all pyrimidinenucleotides that may be present are 2′-deoxy-2′-fluoro nucleotidesexcept for (N N) nucleotides, which can comprise naturally occurringribonucleotides, deoxynucleotides, universal bases, or other chemicalmodifications described herein. The antisense strand of constructs A-Fcomprise sequence complimentary to target RNA sequence of the invention.

FIG. 5 shows non-limiting examples of specific chemically modified siRNAsequences of the invention. A-F applies the chemical modificationsdescribed in FIG. 4A-F to an ADORA1 siRNA sequence.

FIG. 6 shows non-limiting examples of different siRNA constructs of theinvention. The examples shown (constructs 1, 2, and 3) have 19representative base pairs, however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example comprising betweenabout 1, 2, 3, or 4 nucleotides in length, preferably about 2nucleotides. Constructs 1 and 2 can be used independently for RNAiactivity. Construct 2 can comprise a polynucleotide or non-nucleotidelinker, which can optionally be designed as a biodegradable linker. Inone embodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siRNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siRNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siRNA constructs can be modulated based on thedesign of the siRNA construct for use in vivo or in vitro and/or invitro.

FIG. 7 is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate siRNA hairpin constructs.(A) A DNA oligomer is synthesized with a 5′-restriction site (R1)sequence followed by a region having sequence identical (sense region ofsiRNA) to a predetermined ADORA1 target seqeunce, wherein the senseregion comprises, for example, about 19, 20, 21, or 22 nucleotides (N)in length, which is followed by a loop sequence of defined sequence (X),comprising, for example, between about 3 and 10 nucleotides. (B) Thesynthetic construct is then extended by DNA polymerase to generate ahairpin structure having self complementary sequence that will result ina siRNA transcript having specificity for an ADORA1 target sequence andhaving self complementary sense and antisense regions. (C) The constructis heated (for example to about 95° C.) to linearize the sequence, thusallowing extension of a complementary second DNA strand using a primerto the 3′-restriction sequence of the first strand. The double strandedDNA is then inserted into an appropriate vector for expression in cells.The construct can be designed such that a 3′-overhang results from thetranscription, for example by engineering restriction sites and/orutilizing a poly-U termination region as described in Paul et al., 2002,Nature Biotechnology, 29, 505-508.

FIG. 8 is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate double stranded siRNAconstructs. (A) A DNA oligomer is synthesized with a 5′-restriction (R1)site sequence followed by a region having sequence identical (senseregion of siRNA) to a predetermined ADORA1 target seqeunce, wherein thesense region comprises, for example, about 19, 20, 21, or 22 nucleotides(N) in length, and which is followed by a 3′-restriction site (R2) whichis adjacent to a loop sequence of defined sequence (X). (B) Thesynthetic construct is then extended by DNA polymerase to generate ahairpin structure having self complementary sequence. (C) The constructis processed by restriction enzymes specific to R1 and R2 to generate adouble stranded DNA which is then inserted into an appropriate vectorfor expression in cells. The transcription cassette is designed suchthat a U6 promoter region flanks each side of the dsDNA which generatesthe separate sense and antisense strands of the siRNA. Poly Ttermination sequences can be added to the constructs to generate Uoverhangs in the resulting transcript.

FIG. 9 is a diagrammatic representation of a method used to determinetarget sites for siRNA mediated RNAi within a particular target nucleicacid sequence, such as messenger RNA. (A) A pool of siRNAoligonucleotides are synthesized wherein the antisense region of thesiRNA constructs has complementarity to target sites across the targetnucleic acid sequence, and wherein the sense region comprises sequencecomplementary to the antisense region of the siRNA. (B) The sequencesare pooled and are inserted into vectors such that (C) transfection of avector into cells results in the expression of the siRNA. (D) Cells aresorted based on phenotypic change that is associated with modulation ofthe target nucleic acid sequence. (E) The siRNA is isolated from thesorted cells and is sequenced to identify efficacious target siteswithin the target nucleic acid sequence.

MECHANISM OF ACTION OF NUCLEIC ACID MOLECULES OF THE INVENTION

RNA interference refers to the process of sequence specific posttranscriptional gene silencing in animals mediated by short interferingRNAs (siRNA) (Fire et al., 1998, Nature, 391, 806). The correspondingprocess in plants is commonly referred to as post transcriptional genesilencing or RNA silencing and is also referred to as quelling in fungi.The process of post transcriptional gene silencing is thought to be anevolutionarily conserved cellular defense mechanism used to prevent theexpression of foreign genes which is commonly shared by diverse floraand phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protectionfrom foreign gene expression may have evolved in response to theproduction of double stranded RNAs (dsRNA) derived from viral infectionor the random integration of transposon elements into a host genome viaa cellular response that specifically destroys homologous singlestranded RNA or viral genomic RNA. The presence of dsRNA in cellstriggers the RNAi response though a mechanism that has yet to be fullycharacterized. This mechanism appears to be different from theinterferon response that results from dsRNA mediated activation ofprotein kinase PKR and 2′,5′-oligoadenylate synthetase resulting innon-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21-23 nucleotides in length and comprise about 19 base pair duplexes.Dicer has also been implicated in the excision of 21 and 22 nucleotidesmall temporal RNAs (stRNA) from precursor RNA of conserved structurethat are implicated in translational control (Hutvagner et al., 2001,Science, 293, 834). The RNAi response also features an endonucleasecomplex containing a siRNA, commonly referred to as an RNA-inducedsilencing complex (RISC), which mediates cleavage of single stranded RNAhaving sequence homologous to the siRNA. Cleavage of the target RNAtakes place in the middle of the region complementary to the guidesequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15,188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describes RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two nucleotide3′-overhangs. Furthermore, substitution of one or both siRNA strandswith 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity,whereas substitution of 3′-terminal siRNA nucleotides with deoxynucleotides was shown to be tolerated. Mismatch sequences in the centerof the siRNA duplex were also shown to abolish RNAi activity. Inaddition, these studies also indicate that the position of the cleavagesite in the target RNA is defined by the 5′-end of the siRNA guidesequence rather than the 3′-end (Elbashir et al., 2001, EMBO J., 20,6877). Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of a siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA (Nykanen et al., 2001, Cell, 107, 309), however siRNAmolecules lacking a 5′-phosphate are active when introduced exogenously,suggesting that 5′-phosphorylation of siRNA constructs may occur invivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siRNAoligonucleotide sequences or siRNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45sec coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table II outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick &Jackson Synthesis Grade acetonitrile is used directly from the reagentbottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made upfrom the solid obtained from American International Chemical, Inc.Alternately, for the introduction of phosphorothioate linkages, Beaucagereagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile)is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aq. methylamine(1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatantis removed from the polymer support. The support is washed three timeswith 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is thenadded to the first supernatant. The combined supernatants, containingthe oligoribonucleotide, are dried to a white powder.

The method of synthesis used for RNA including certain siRNA moleculesof the invention follows the procedure as described in Usman et al.,1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic AcidsRes., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23,2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes useof common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 7.5 min coupling step for alkylsilyl protected nucleotides and a2.5 min coupling step for 2′-O-methylated nucleotides. Table II outlinesthe amounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mMI₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & JacksonSynthesis Grade acetonitrile is used directly from the reagent bottle.S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from thesolid obtained from American International Chemical, Inc. Alternately,for the introduction of phosphorothioate linkages, Beaucage reagent(3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL)at 65° C. for 15 min. The vial is brought to r.t. TEA.3HF (0.1 mL) isadded and the vial is heated at 65° C. for 15 min. The sample is cooledat −20° C. and then quenched with 1.5 M NH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 min. The cartridge is then washed again with water, salt exchangedwith 1 M NaCl and washed with water again. The oligonucleotide is theneluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format, all that is important is the ratio ofchemicals used in the reaction.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siRNA molecules of the invention can also be synthesized via atandem synthesis methodology as described in Example 1 herein, whereinboth siRNA strands are synthesized as a single contiguousoligonucleotide fragment or strand separated by a cleavable linker whichis subsequently cleaved to provide separate siRNA fragments or strandsthat hybridize and permit purification of the siRNA duplex. The linkercan be a polynucleotide linker or a non-nucleotide linker. The tandemsynthesis of siRNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siRNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

A siRNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siRNA constructs can bepurified by gel electrophoresis using general methods or can be purifiedby high pressure liquid chromatography (HPLC; see Wincott et al., supra,the totality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siRNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siRNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siRNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siRNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro,2′-O-methyl, 2′-O-allyl, 2-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siRNA nucleic acid moleculesof the instant invention so long as the ability of siRNA to promote RNAiis cells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorothioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Small interfering RNA (siRNA) molecules having chemical modificationsthat maintain or enhance activity are provided. Such a nucleic acid isalso generally more resistant to nucleases than an unmodified nucleicacid. Accordingly, the in vitro and/or in vivo activity should not besignificantly lowered. In cases in which modulation is the goal,therapeutic nucleic acid molecules delivered exogenously shouldoptimally be stable within cells until translation of the target RNA hasbeen modulated long enough to reduce the levels of the undesirableprotein. This period of time varies between hours to days depending uponthe disease state. Improvements in the chemical synthesis of RNA and DNA(Wincott et al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al.,1992, Methods in Enzymology 211,3-19 (incorporated by reference herein))have expanded the ability to modify nucleic acid molecules byintroducing nucleotide modifications to enhance their nucleasestability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siRNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siRNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,phospholipids, nucleosides, nucleotides, nucleic acids, antibodies,toxins, negatively charged polymers and other polymers, for exampleproteins, peptides, hormones, carbohydrates, polyethylene glycols, orpolyamines, across cellular membranes. In general, the transportersdescribed are designed to be used either individually or as part of amulti-component system, with or without degradable linkers. Thesecompounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to a siRNA molecule of the invention or thesense and antisense strands of a siRNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein, refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siRNA molecules either alone or in combination with othemolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, hormones, antivirals, peptides,proteins, chemotherapeutics, small molecules, vitamins, co-factors,nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids,antisense nucleic acids, triplex forming oligonucleotides, 2,5-Achimeras, siRNA, dsRNA, allozymes, aptamers, decoys and analogs thereofBiologically active molecules of the invention also include moleculescapable of modulating the pharmacokinetics and/or pharmacodynamics ofother biologically active molecules, for example, lipids and polymerssuch as polyamines, polyamides, polyethylene glycol and otherpolyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siRNA molecules) deliveredexogenously optimally are stable within cells until reverse trascriptionof the RNA has been modulated long enough to reduce the levels of theRNA transcript. The nucleic acid molecules are resistant to nucleases inorder to function as effective intracellular therapeutic agents.Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siRNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siRNA molecules targeted todifferent genes; nucleic acid molecules coupled with known smallmolecule modulators; or intermittent treatment with combinations ofmolecules, including different motifs and/or other chemical orbiological molecules). The treatment of subjects with siRNA moleculescan also include combinations of different types of nucleic acidmolecules, such as enzymatic nucleic acid molecules (ribozymes),allozymes, antisense, 2,5-A oligoadenylate, decoys, aptamers etc.

In another aspect a siRNA molecule of the invention comprises one ormore 5′ and/or a 3′-cap structure, for example on only the sense siRNAstrand, antisense siRNA strand, or both siRNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples: the 5′-cap is selected from the groupcomprising inverted abasic residue (moiety); 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety.

In yet another preferred embodiment, the 3′-cap is selected from a groupcomprising, 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkylphosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate;6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropylphosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;alpha-nucleotide; modified base nucleotide; phosphorodithioate;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide;3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety;5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate;5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group canbe substituted or unsubstituted. When substituted the substitutedgroup(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, or SH. The term also includes alkenyl groups that are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkenyl group has 1 to 12 carbons. More preferably, it is a loweralkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups that have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO2 or N(CH₃)₂, amino orSH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo,pyrimidyl, pyrazinyl, imidazolyl and the like, all optionallysubstituted. An “amide” refers to an —C(O)—NH—R, where R is eitheralkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′,where R is either alkyl, aryl, alkylaryl or hydrogen.

By “nucleotide” as used herein is as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified siRNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, and Mesmaeker et al., 1994, Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, uracil joined to the 1′ carbon of β-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which may be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878, which are both incorporated by reference in theirentireties.

Various modifications to nucleic acid siRNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

A siRNA molecule of the invention can be adapted for use to treat, forexample allergic/inflammatory diseases and conditions, including but notlimited to asthma, allergic rhinitis, atopic dermatitis, and any otherindications that can respond to the level of ADORA1 in a cell or tissue,alone or in combination with other therapies. For example, a siRNAmolecule can comprise a delivery vehicle, including liposomes, foradministration to a subject, carriers and diluents and their salts,and/or can be present in pharmaceutically acceptable formulations.Methods for the delivery of nucleic acid molecules are described inAkhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies forAntisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al.,1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb.Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser.,752, 184-192, all of which are incorporated herein by reference.Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO94/02595, further describes the general methods for delivery of nucleicacid molecules. These protocols can be utilized for the delivery ofvirtually any nucleic acid molecule. Nucleic acid molecules can beadministered to cells by a variety of methods known to those of skill inthe art, including, but not restricted to, encapsulation in liposomes,by iontophoresis, or by incorporation into other delivery vehicles, suchas hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). Alternatively, thenucleic acid/vehicle combination is locally delivered by directinjection or by use of an infusion pump. Direct injection of the nucleicacid molecules of the invention, whether subcutaneous, intramuscular, orintradermal, can take place using standard needle and syringemethodologies, or by needle-free technologies such as those described inConry et al., 1999, Clin. Cancer Res., 5, 2330-2337 and Barry et al.,International PCT Publication No. WO 99/31262. Many examples in the artdescribe CNS delivery methods of oligonucleotides by osmotic pump, (seeChun et al., 1998, Neuroscience Letters, 257, 135-138, D'Aldin et al.,1998, Mol. Brain Research, 55, 151-164, Dryden et al., 1998, J.

Endocrinol., 157, 169-175, Ghirnikar et al., 1998, Neuroscience Letters,247, 21-24) or direct infusion (Broaddus et al., 1997, Neurosurg. Focus,3, article 4). Other routes of delivery include, but are not limited tooral (tablet or pill form) and/or intrathecal delivery (Gold, 1997,Neuroscience, 76, 1153-1158). More detailed descriptions of nucleic aciddelivery and administration are provided in Sullivan et al., supra,Draper et al., PCT WO93/23569, Beigelman et al., PCT WO99/05094, andKlimuk et al., PCT WO99/04819 all of which have been incorporated byreference herein.

In addition, the invention features the use of methods to deliver thenucleic acid molecules of the instant invention to hematopoietic cells,including monocytes and lymphocytes. These methods are described indetail by Hartmann et al., 1998, J. Phamacol. Exp. Ther., 285(2),920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862; Filion andPhillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei,1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al., 1994, NucleicAcids Research, 22(22), 4681-8. Such methods, as described above,include the use of free oligonucleitide, cationic lipid formulations,liposome formulations including pH sensitive liposomes andimmunoliposomes, and bioconjugates including oligonucleotides conjugatedto fusogenic peptides, for the transfection of hematopoietic cells witholigonucleotides.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedinto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention may also be formulated and used as tablets, capsules orelixirs for oral administration, suppositories for rectaladministration, sterile solutions, suspensions for injectableadministration, and the other compositions known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or subject, including for example a human.Suitable forms, in part, depend upon the use or the route of entry, forexample oral, transdermal, or by injection. Such forms should notprevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption oraccumulation of drugs in the blood stream followed by distributionthroughout the entire body. Administration routes which lead to systemicabsorption include, without limitation: intravenous, subcutaneous,intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.Each of these administration routes expose the siRNA molecules of theinvention to an accessible diseased tissue. The rate of entry of a druginto the circulation has been shown to be a function of molecular weightor size. The use of a liposome or other drug carrier comprising thecompounds of the instant invention can potentially localize the drug,for example, in certain tissue types, such as the tissues of thereticular endothelial system (RES). A liposome formulation that canfacilitate the association of drug with the surface of cells, such as,lymphocytes and macrophages is also useful. This approach may provideenhanced delivery of the drug to target cells by taking advantage of thespecificity of macrophage and lymphocyte immune recognition of abnormalcells, such as cancer cells.

By “pharmaceutically acceptable formulation” is meant, a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity. Non-limiting examples of agentssuitable for formulation with the nucleic acid molecules of the instantinvention include the forulations and conjugates described herein, aswell as other target area specific formulations including CNSformulations including P-glycoprotein inhibitors (such as Pluronic P85),which can enhance entry of drugs into the CNS (Jolliet-Riant andTillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery after intracerebral implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge,Mass.); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate, which can deliver drugs across the blood brainbarrier and can alter neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Othernon-limiting examples of delivery strategies for the nucleic acidmolecules of the instant invention include material described in Boadoet al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBSLett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596;Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada etal., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999,PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995,95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al.,1995,Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage oradministration, which include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985)hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents may be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono-or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention may also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication may increase the beneficialeffects while reducing the presence of side effects.

In one embodiment, the invention compositions suitable for administeringnucleic acid molecules of the invention to specific cell types. Forexample, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J.Biol. Chem. 262, 4429-4432) is unique to hepatocytes and binds branchedgalactose-terminal glycoproteins, such as asialoorosomucoid (ASOR).Binding of such glycoproteins or synthetic glycoconjugates to thereceptor takes place with an affinity that strongly depends on thedegree of branching of the oligosaccharide chain, for example,triatennary structures are bound with greater affinity than biatenarryor monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620;Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987,Glycoconjugate J., 4, 317-328, obtained this high specificity throughthe use of N-acetyl-D-galactosamine as the carbohydrate moiety, whichhas higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose andgalactosamine based conjugates to transport exogenous compounds acrosscell membranes can provide a targeted delivery approach to the treatmentof liver disease or hepatocellular carcinoma. The use of bioconjugatescan also provide a reduction in the required dose of therapeuticcompounds required for treatment. Furthermore, therapeuticbioavialability, pharmacodynamics, and pharmacokinetic parameters can bemodulated through the use of nucleic acid bioconjugates of theinvention. Non-limiting examples of such bioconjugates are described inVargeese et al., U.S. Ser. No. 60/311,865, filed Aug. 13, 2001; andMatulic-Adamic et al., U.S. Ser. No. 60/362,016, filed Mar. 6, 2002.

Alternatively, certain siRNA molecules of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad.Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev.,2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe etal., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad.Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20,4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al.,1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4,45. Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector. Theactivity of such nucleic acids can be augmented by their release fromthe primary transcript by a enzymatic nucleic acid (Draper et al., PCTWO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992,Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic AcidsRes., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21,3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siRNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Thompson, U.S. Pat.Nos. 5,902,880 and 6,146,886). The recombinant vectors capable ofexpressing the siRNA molecules can be delivered as described above, andpersist in target cells. Alternatively, viral vectors can be used thatprovide for transient expression of nucleic acid molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siRNAmolecule interacts with the target mRNA and generates an RNAi response.Delivery of siRNA molecule expressing vectors can be systemic, such asby intravenous or intra-muscular administration, by administration totarget cells ex-planted from a subject followed by reintroduction intothe subject, or by any other means that would allow for introductioninto the desired target cell (for a review see Couture et al., 1996,TIG., 12, 510).

In one aspect the invention features an expression vector comprising anucleic acid sequence encoding at least one siRNA molecule of theinstant invention. The expression vector can encode one or both strandsof a siRNA duplex, or a single self complementary strand that selfhybridizes into a siRNA duplex. The nucleic acid sequences encoding thesiRNA molecules of the instant invention can be operably linked in amanner that allows expression of the siRNA molecule (see for examplePaul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira,2002, Nature Biotechnology, 19, 497; Lee et al., 2002, NatureBiotechnology, 19, 500; and Novina et al., 2002, Nature Medicine,advance online publication doi:10.1038/nm725).

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); and c) a nucleicacid sequence encoding at least one of the siRNA molecules of theinstant invention; wherein said sequence is operably linked to saidinitiation region and said termination region, in a manner that allowsexpression and/or delivery of the siRNA molecule. The vector canoptionally include an open reading frame (ORF) for a protein operablylinked on the 5′ side or the 3′-side of the sequence encoding the siRNAof the invention; and/or an intron (intervening sequences).

Transcription of the siRNA molecule sequences can be driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol II promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gaoand Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993,Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10,4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al.,1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S.A, 90, 8000-4;Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,1993, Science, 262, 1566). More specifically, transcription units suchas the ones derived from genes encoding U6 small nuclear (snRNA),transfer RNA (tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siRNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al.,International PCT Publication No. WO 96/18736. The above siRNAtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprisinga nucleic acid sequence encoding at least one of the siRNA molecules ofthe invention, in a manner that allows expression of that siRNAmolecule. The expression vector comprises in one embodiment; a) atranscription initiation region; b) a transcription termination region;and c) a nucleic acid sequence encoding at least one strand of the siRNAmolecule; wherein the sequence is operably linked to the initiationregion and the termination region, in a manner that allows expressionand/or delivery of the siRNA molecule.

In another embodiment the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; and d) a nucleic acid sequence encoding atleast one strand of a siRNA molecule, wherein the sequence is operablylinked to the 3′-end of the open reading frame; and wherein the sequenceis operably linked to the initiation region, the open reading frame andthe termination region, in a manner that allows expression and/ordelivery of the siRNA molecule. In yet another embodiment the expressionvector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; and d) a nucleic acidsequence encoding at least one siRNA molecule; wherein the sequence isoperably linked to the initiation region, the intron and the terminationregion, in a manner which allows expression and/or delivery of thenucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; and e) a nucleic acid sequenceencoding at least one strand of a siRNA molecule, wherein the sequenceis operably linked to the 3′-end of the open reading frame; and whereinthe sequence is operably linked to the initiation region, the intron,the open reading frame and the termination region, in a manner whichallows expression and/or delivery of the siRNA molecule.

Examples

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1 Tandem Synthesis of siRNA Constructs

Exemplary siRNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example a succinyl-based linker. Tandemsynthesis as described herein is followed by a one step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siRNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of an siRNA oligo and its complimentin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove. Following deprotection, the siRNA sequence strands are allowed tospontaneously hybridize. This hybridization yields a duplex in which onestrand has retained the 5′-O-DMT group while the complementary strandcomprises a terminal 5′-hydroxyl. The newly formed duplex to behaves asa single molecule during routine solid-phase extraction purification(Trityl-On purification) even though only one molecule has adimethoxytrityl group. Because the strands form a stable duplex, thisdimethoxytrityl group (or an equivalent group, such as other tritylgroups or other hydrophobic moieties) is all that is required to purifythe pair of oligos, for example by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to point ofintroducing a tandem linker, such as an inverted deoxyabasic succinatelinker (see FIG. 1) or an equivalent cleavable linker. A non-limitingexample of linker coupling conditions that can be used includes ahindered base such as diisopropylethylamine (DIPA) and/or DMAP in thepresence of an activator reagent such asBromotripyrrolidinophosphoniumhexaflurorophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siRNA duplex can be readily accomplished using solidphase extraction, for example using a Waters C18 SepPak 1 g cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H2O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapprox. 10 minutes. The remaining TFA solution is removed and the columnwashed with H2O followed by 1 CV 1M NaCl and additional H2O. The siRNAduplex product is then eluted, for example using 1 CV 20% aqueous CAN.

FIG. 2 provides an example of MALDI-TOV mass spectrometry analysis of apurified siRNA construct in which each peak corresponds to thecalculated mass of an individual siRNA strand of the siRNA duplex. Thesame purified siRNA provides three peaks when analyzed by capillary gelelectrophoresis (CGE), one peak presumably corresponding to the duplexsiRNA, and two peaks presumably corresponding to the separate siRNAsequence strands. Ion exchange HPLC analysis of the same siRNA contractonly shows a single peak.

Example 2 Identification of Potential siRNA Target Sites in Any RNASequence

The sequence of an RNA target of interest, such as a human mRNAtranscript, is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siRNA targets having complimentarity to the target.Such sequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease or condition such as those sites containing mutations ordeletions, can be used to design siRNA molecules targeting those sitesas well. Various parameters can be used to determine which sites are themost suitable target sites within the target RNA sequence. Theseparameters include but are not limited to secondary or tertiary RNAstructure, the nucleotide base composition of the target sequence, thedegree of homology between various regions of the target sequence, orthe relative position of the target sequence within the RNA transcript.Based on these determinations, any number of target sites within the RNAtranscript can be chosen to screen siRNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. In a non-limiting example, anywhere from 1 to 1000 target sitesare chosen within the transcript based on the size of the siRNAconstruct to be used. High throughput screening assays can be developedfor screening siRNA molecules using methods known in the art, such aswith multi-well or multi-plate assays to determine efficient reductionin target gene expression.

Example 3 Selection of siRNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selectionof siRNAs targeting a given gene sequence or transcript.

-   1. The target sequence is parsed in silico into a list of all    fragments or subsequences of a particular length, for example 23    nucleotide fragments, contained within the target sequence. This    step is typically carried out using a custom Perl script, but    commercial sequence analysis programs such as Oligo, MacVector, or    the GCG Wisconsin Package can be employed as well.-   2. In some instances the siRNAs correspond to more than one target    sequence; such would be the case for example in targeting different    transcripts of the same gene, targeting different transcripts of    more than one gene, or for targeting both the human gene and an    animal homolog. In this case, a subsequence list of a particular    length is generated for each of the targets, and then the lists are    compared to find matching sequences in each list. The subsequences    are then ranked according to the number of target sequences that    contain the given subsequence; the goal is to find subsequences that    are present in most or all of the target sequences. Alternately, the    ranking can identify subsequences that are unique to a target    sequence, such as a mutant target sequence. Such an approach would    enable the use of siRNA to target specifically the mutant sequence    and not effect the expression of the normal sequence.-   3. In some instances the siRNA subsequences are absent in one or    more sequences while present in the desired target sequence; such    would be the case if the siRNA targets a gene with a paralogous    family member that is to remain untargeted. As in case 2 above, a    subsequence list of a particular length is generated for each of the    targets, and then the lists are compared to find sequences that are    present in the target gene but are absent in the untargeted paralog.-   4. The ranked siRNA subsequences can be further analyzed and ranked    according to GC content. A preference can be given to sites    containing 30-70% GC, with a further preference to sites containing    40-60% GC.-   5. The ranked siRNA subsequences can be further analyzed and ranked    according to self-folding and internal hairpins. Weaker internal    folds are preferred; strong hairpin structures are to be avoided.-   6. The ranked siRNA subsequences can be further analyzed and ranked    according to whether they have runs of GGG or CCC in the sequence.    GGG (or even more Gs) in either strand can make oligonucleotide    synthesis problematic, so it is avoided whenever better sequences    are available. CCC is searched in the target strand because that    will place GGG in the antisense strand.-   7. The ranked siRNA subsequences can be further analyzed and ranked    according to whether they have the dinucleotide UU (uridine    dinucleotide) on the 3′ end of the sequence, and/or AA on the 5′ end    of the sequence (to yield 3′ UU on the antisense sequence). These    sequences allow one to design siRNA molecules with terminal TT    thymidine dinucleotides.-   8. Four or five target sites are chosen from the ranked list of    subsequences as described above. For example, in subsequences having    23 nucleotides, the right 21 nucleotides of each chosen 23-mer    subsequence are then designed and synthesized for the upper (sense)    strand of the siRNA duplex, while the reverse complement of the left    21 nucleotides of each chosen 23-mer subsequence are then designed    and synthesized for the lower (antisense) strand of the siRNA    duplex. If terminal TT residues are desired for the sequence (as    described in paragraph 7), then the two 3′ terminal nucleotides of    both the sense and antisense strands are replaced by TT prior to    synthesizing the oligos.-   9. The siRNA molecules are screened in an in vitro, cell culture or    animal model system to identify the most active siRNA molecule or    the most preferred target site within the target RNA sequence.

In an alternate approach, a pool of siRNA constructs specific to anADORA1 target sequence is used to screen for target sites in cellsexpressing ADORA1 RNA, such as human lung mast cells. The generalstrategy used in this approach is shown in FIG. 9. A non-limitingexample of such as pool is a pool comprising sequences having sensesequences comprising SEQ ID NOs. 1-161 and antisense sequencescomprising SEQ ID NOs. 162-322 respectively. Human lung mast cellsexpressing ADORA1 are transfected with the pool of siRNA constructs andcells that demonstrate a phenotype associated with ADORA1 inhibition aresorted. The pool of siRNA constructs can be expressed from transciptioncassettes inserted into appropriate vectors (see for example FIG. 7 andFIG. 8). The siRNA from cells demonstrating a positive phenotypic change(e.g., decreased adenosine receptor expression, for example asdetermined by a [³H]DPCPX binding assay as described in Nyce andMetzger, 1997, Nature, 385, 721-725), are sequenced to determine themost suitable target site(s) within the target ADORA1 RNA sequence.

Example 4 ADORA1 Targeted siRNA Design

siRNA target sites were chosen by analyzing sequences of the ADORA1 RNAtarget and optionally prioritizing the target sites on the basis offolding (structure of any given sequence analyzed to determine siRNAaccessibility to the target), using a library of siRNA molecules asdescribed in Example 3, or alternately by using an in vitro siRNA systemas described in Example 6 herein. siRNA molecules were designed thatcould bind each target and are optionally individually analyzed bycomputer folding to assess whether the siRNA molecule can interact withthe target sequence. Varying the length of the siRNA molecules can bechosen to optimize activity. Generally, a sufficient number ofcomplimentary nucleotide bases are chosen to bind to, or otherwiseinteract with, the target RNA, but the degree of complementarity can bemodulated to accommodate siRNA duplexes or varying length or basecomposition. By using such methodologies, siRNA molecules can bedesigned to target sites within any known RNA sequence, for examplethose RNA sequences corresponding to the any gene transcript.

Example 5 Chemical Synthesis and Purification of siRNA

siRNA molecules can be designed to interact with various sites in theRNA message, for example target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siRNA molecule(s)are complementary to the target site sequences described above. ThesiRNA molecules can be chemically synthesized using methods describedherein. Inactive siRNA molecules that are used as control sequences canbe synthesized by scrambling the sequence of the siRNA molecules suchthat it is not complimentary to the target sequence.

Example 6 RNAi in vitro Assay to Assess siRNA Activity

An in vitro assay that recapitulates RNAi in a cell free system is usedto evaluate siRNA constructs targeting ADORA1 RNA targets. The assaycomprises the system described by Tuschl et al., 1999, Genes andDevelopment, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33adapted for use with ADORA1 target RNA. A Drosophila extract derivedfrom syncytial blastoderm is used to reconstitute RNAi activity invitro. Target RNA is generated via in vitro transcription from anappropriate ADORA1 expressing plasmid using T7 RNA polymerase or viachemical synthesis as described herein. Sense and antisense siRNAstrands (for example 20 uM each) are annealed by incubation in buffer(such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mMmagnesium acetate) for 1 min. at 90° C. followed by 1 hour at 37° C. ,then diluted in lysis buffer (for example 100 mM potassium acetate, 30mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can bemonitored by gel electrophoresis on an agarose gel in TBE buffer andstained with ethidium bromide. The Drosophila lysate is prepared usingzero to two hour old embryos from Oregon R flies collected on yeastedmolasses agar that are dechorionated and lysed. The lysate iscentrifuged and the supernatant isolated. The assay comprises a reactionmixture containing 50% lysate [vol/vol], RNA (10-50 pM finalconcentration), and 10% [vol/vol] lysis buffer containing siRNA (10 nMfinal concentration). The reaction mixture also contains 10 mM creatinephosphate, 10 ug/ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM ofeach amino acid. The final concentration of potassium acetate isadjusted to 100 mM. The reactions are pre-assembled on ice andpreincubated at 25° C. for 10 minutes before adding RNA, then incubatedat 25° C. for an additional 60 minutes. Reactions are quenched with 4volumes of 1.25× Passive Lysis Buffer (Promega). Target RNA cleavage isassayed by RT-PCR analysis or other methods known in the art and arecompared to control reactions in which siRNA is omitted from thereaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [a-³²P] CTP, passed over a G50 Sephadex column by spin chromatography and used as target RNA withoutfurther purification. Optionally, target RNA is 5′-³²P-end labeled usingT4 polynucleotide kinase enzyme. Assays are performed as described aboveand target RNA and the specific RNA cleavage products generated by RNAiare visualized on an autoradiograph of a gel. The percentage of cleavageis determined by Phosphor Imager® quantitation of bands representingintact control RNA or RNA from control reactions without siRNA and thecleavage products generated by the assay.

In one embodiment, this assay is used to determine target sites theADORA1 RNA target for siRNA mediated RNAi cleavage, wherein a pluralityof siRNA constructs are screened for RNAi mediated cleavage of theADORA1 RNA target, for example by analyzing the assay reaction byelectrophoresis of labeled target RNA, or by northern blotting, as wellas by other methodology well known in the art.

Example 7 Nucleic Acid Onhibition of ADORA1 Target RNA in vivo

siRNA molecules targeted to the human ADORA1 RNA are designed andsynthesized as described above. These nucleic acid molecules can betested for cleavage activity in vivo, for example, using the followingprocedure. The target sequences and the nucleotide location within theADORA1 RNA are given in Table I and III.

Two formats are used to test the efficacy of siRNAs targeting ADORA1.First, the reagents are tested on human lung epithelial cells (e.g.,A549), to determine the extent of RNA and protein inhibition. siRNAreagents (e.g.; see Table I, and III) are selected against the ADORA1target. RNA inhibition is measured after delivery of these reagents by asuitable transfection agent to human lung epithelial cells. Relativeamounts of target RNA are measured versus actin using real-time PCRmonitoring of amplification (eg. ABI 7700 Taqman®). A comparison is madeto a mixture of oligonucleotide sequences made to unrelated targets orto a randomized siRNA control with the same overall length andchemistry, but randomly substituted at each position. Primary andsecondary lead reagents are chosen for the target and optimizationperformed. After an optimal transfection agent concentration is chosen,a RNA time-course of inhibition is performed with the lead siRNAmolecule. In addition, a cell-plating format can be used to determineRNA inhibition.

Delivery of siRNA to Lung Epithelial Cells

Human lung epithelial cells (e.g., A549) are seeded, for example, at1×10⁵ cells per well of a six well dish in EGM-2 (BioWhittaker) the daybefore transfection. siRNA (final concentration, for example 20 nM) andcationic lipid (e.g., final concentration 2 μg/ml) are complexed in EGMbasal media (Biowhittaker) at 37° C. for 30 mins in polystyrene tubes.Following vortexing, the complexed siRNA is added to each well andincubated for the times indicated. For initial optimization experiments,cells are seeded, for example, at 1×10³ in 96 well plates and siRNAcomplex added as described. Efficiency of delivery of siRNA to A549 isdetermined using a fluorescent siRNA complexed with lipid. A549 in 6well dishes are incubated with siRNA for 24 hours, rinsed with PBS andfixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptakeof siRNA is visualized using a fluorescent microscope.

Taqman and Lightcycler Quantification of mRNA

Total RNA is prepared from cells following siRNA delivery, for exampleusing Qiagen RNA purification kits for 6 well or Rneasy extraction kitsfor 96 well assays. For Taqman analysis, dual-labeled probes aresynthesized with the reporter dye, FAM or JOE, covalently linked at the5′ end and the quencher dye TAMRA conjugated to the 3′ end. One-stepRT-PCR amplifications are performed on, for example, an ABI PRISM 7700Sequence Detector using 50 μl reactions consisting of 10 μl total RNA,100 nM forward primer, 900 nM reverse primer, 100 nM probe, 1× TaqManPCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM eachdATP, dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega), 1.25UAmpliTaq Gold (PE-Applied Biosystems) and 10U M-MLV ReverseTranscriptase (Promega). The thermal cycling conditions can consist of30 min at 48° C., 10 min at 95° C., followed by 40 cycles of 15 sec at95° C. and 1 min at 60° C. Quantitation of mRNA levels are determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/rxn) and normalizing to β-actin or GAPDH mRNA inparallel TaqMan reactions. For each gene of interest an upper and lowerprimer and a flourescently labeled probe are designed. Real timeincorporation of SYBR Green I dye into a specific PCR product can bemeasured in glass capillary tubes using a lightcyler. A standard curveis generated for each primer pair using control c RNA allularnd valuesare represented as relative expression to GAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micropreparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 8 Models Useful to Evaluate the Down-Regulation of ADORA1 GeneExpression Animal Models

Evaluating the efficacy of anti-ADORA-1 agents (e.g., siRNA) in animalmodels is an important prerequisite to human clinical trials. Nyce andMetzger, 1997, Nature, 385, 721-725, describe a useful dust miteconditioned allergic rabbit model of human asthma. Allergic rabbitstreated with aerosolized siRNA are compared to untreated controls oranimals treated with a non-specific siRNA control with regard toadenosine challenge. The concentration of aerolsolized adenosinerequired to reduce the dynamic compliance of the bronchial airway 50%from a baseline values is determined in both groups of animals.Additionally, dose response studies using this same endpoint areperformed. Airway smooth muscle is surgically dissected from the animalsand is processed for quantitative assessment of adenosine A1 receptors.As a control for specificity, adenosine A2 receptors and/or bradykininreceptors are quantitated as well. Adenosine A1 receptor density can beassayed by specific binding of a [³H]DPCPX. A dose dependent reductionin adenosine A1 receptor density is indicative of a therapeutic responseThis model can be used to evaluate animals that are treated with nucleicacid molecules of the invention and can furthermore be used as apositive control in determining the response of animals treated withnucleic acid molecules of the invention by using such factors as airwayobstruction, lung capacity, and bronchiolar alveolar lavage (BAL) fluidin the evaluation.

Cell Culture

Human epithelial lung cell lines, such as NPE cells and NCB-20 cells,can be used to express ADORA1. Cloned human ADORA1 is thereforeexpressed in CHO and COST cells and used in various studies. TheseADORA1 expressing lung cell lines can be used in cell culture assays toevaluate nucleic acid molecules of the invention. A primary endpoint inthese experiments would be the RT-PCR analysis of ADORA1 mRNA expressionin ADORA1 expressing cells. In addition, ligand binding assays can bedeveloped where binding of [³H]DPCPX can be evaluated in response totreatment with nucleic acid molecules of the invention.

Example 9 Indications

The present body of knowledge in ADORA1 research indicates the need formethods to assay ADORA1 activity and for compounds that can regulateADORA1 expression for research, diagnostic, and therapeutic use. Asdescribed herein, the nucleic acid molecules of the present inventioncan be used in assays to diagnose disease state related of ADORA1levels. In addition, the nucleic acid molecules can be used to treatdisease state related to ADORA1 levels.

Particular degenerative and disease states that can be associated withADORA1 levels include, but are not limited to allergic diseases andconditions, including but not limited to asthma, allergic rhinitis,atopic dermatitis, and any other diseases or conditions that are relatedto or will respond to the levels of ADORA1 in a cell or tissue, alone orin combination with other therapies.

The use of anti-inflammatories, bronchodilators, adenosine inhibitorsand adenosine A1 receptor inhibitors are examples of other treatments ortherapies can be combined with the nucleic acid molecules of theinvention. Those skilled in the art will recognize that other drugcompounds and therapies can be similarly be readily combined with thenucleic acid molecules of the instant invention (e.g., siRNA molecules)are hence within the scope of the instant invention.

Example 10 Diagnostic Uses

The siRNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in identifying molecular targets suchas RNA in a variety of applications, for example, in clinical,industrial, environmental, agricultural and/or research settings. Suchdiagnostic use of siRNA molecules involves utilizing reconstituted RNAisystems, for example using cellular lysates or partially purifiedcellular lysates. siRNA molecules of this invention may be used asdiagnostic tools to examine genetic drift and mutations within diseasedcells or to detect the presence of endogenous or exogenous, for exampleviral, RNA in a cell. The close relationship between siRNA activity andthe structure of the target RNA allows the detection of mutations in anyregion of the molecule, which alters the base-pairing andthree-dimensional structure of the target RNA. By using multiple siRNAmolecules described in this invention, one may map nucleotide changes,which are important to RNA structure and function in vitro, as well asin cells and tissues. Cleavage of target RNAs with siRNA molecules canbe used to inhibit gene expression and define the role (essentially) ofspecified gene products in the progression of disease or infection. Inthis manner, other genetic targets may be defined as important mediatorsof the disease. These experiments will lead to better treatment of thedisease progression by affording the possibility of combinationtherapies (e.g., multiple siRNA molecules targeted to different genes,siRNA molecules coupled with known small molecule inhibitors, orintermittent treatment with combinations siRNA molecules and/or otherchemical or biological molecules). Other in vitro uses of siRNAmolecules of this invention are well known in the art, and includedetection of the presence of mRNAs associated with a disease, infection,or related condition. Such RNA is detected by determining the presenceof a cleavage product after treatment with a siRNA using standardmethodologies, for example fluorescence resonance emission transfer(FRET).

In a specific example, siRNA molecules that can cleave only wild-type ormutant forms of the target RNA are used for the assay. The first siRNAmolecules is used to identify wild-type RNA present in the sample andthe second siRNA molecules will be used to identify mutant RNA in thesample. As reaction controls, synthetic substrates of both wild-type andmutant RNA will be cleaved by both siRNA molecules to demonstrate therelative siRNA efficiencies in the reactions and the absence of cleavageof the “non-targeted” RNA species. The cleavage products from thesynthetic substrates will also serve to generate size markers for theanalysis of wild-type and mutant RNAs in the sample population. Thuseach analysis will require two siRNA molecules, two substrates and oneunknown sample which will be combined into six reactions. The presenceof cleavage products will be determined using an RNase protection assayso that full-length and cleavage fragments of each RNA can be analyzedin one lane of a polyacrylamide gel. It is not absolutely required toquantify the results to gain insight into the expression of mutant RNAsand putative risk of the desired phenotypic changes in target cells. Theexpression of mRNA whose protein product is implicated in thedevelopment of the phenotype (i.e., disease related or infectionrelated) is adequate to establish risk. If probes of comparable specificactivity are used for both transcripts, then a qualitative comparison ofRNA levels will be adequate and will decrease the cost of the initialdiagnosis. Higher mutant form to wild-type ratios will be correlatedwith higher risk whether RNA levels are compared qualitatively orquantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments, optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the description and the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I ADORA1 target and siRNA sequences (5′-3′) Seq Seq Seq PosTarget Sequence ID UPos Upper seq ID LPos Lower seq ID 3GAGUGUCAGAAGUGUGAAG 1 3 GAGUGUCAGAAGUGUGAAG 1 21 CUUCACACUUCUGACACUC 16221 GGGUGCCUGUUCUGAAUCC 2 21 GGGUGCCUGUUCUGAAUCC 2 39 GGAUUCAGAACAGGCACCC163 39 CCAGAGCCUCCUCUCCCUC 3 39 CCAGAGCCUCCUCUCCCUC 3 57GAGGGAGAGGAGGCUCUGG 164 57 CUGUGAGGCUGGCAGGUGA 4 57 CUGUGAGGCUGGCAGGUGA4 75 UCACCUGCCAGCCUCACAG 165 75 AGGAAGGGUUUAACCUCAC 5 75AGGAAGGGUUUAACCUCAC 5 93 GUGAGGUUAAACCCUUCCU 166 93 CUGGAAGGAAUCCCUGGAG6 93 CUGGAAGGAAUCCCUGGAG 6 111 CUCCAGGGAUUCCUUCCAG 167 111GCUAGCGGCUGCUGAAGGC 7 111 GCUAGCGGCUGCUGAAGGC 7 129 GCCUUCAGCAGCCGCUAGC168 129 CGUCGAGGUGUGGGGGCAC 8 129 CGUCGAGGUGUGGGGGCAC 8 147GUGCCCCCACACCUCGACG 169 147 CUUGGACAGAACAGUCAGG 9 147CUUGGACAGAACAGUCAGG 9 165 CCUGACUGUUCUGUCCAAG 170 165GCAGCCGGGAGCUCUGCCA 10 165 GCAGCCGGGAGCUCUGCCA 10 183UGGCAGAGCUCCCGGCUGC 171 183 AGCUUUGGUGACCUUGGGC 11 183AGCUUUGGUGACCUUGGGC 11 201 GCCCAAGGUCACCAAAGCU 172 201CCGGGCUGGGAGCGCUGCG 12 201 CCGGGCUGGGAGCGCUGCG 12 219CGCAGCGCUCCCAGCCCGG 173 219 GGCGGGAGCCGGAGGACUA 13 219GGCGGGAGCCGGAGGACUA 13 237 UAGUCCUCCGGCUCCCGCC 174 237AUGAGCUGCCGCGCGUUGU 14 237 AUGAGCUGCCGCGCGUUGU 14 255ACAACGCGCGGCAGCUCAU 175 255 UCCAGAGCCCAGCCCAGCC 15 255UCCAGAGCCCAGCCCAGCC 15 273 GGCUGGGCUGGGCUCUGGA 176 273CCUACGCGCGCGGCCCGGA 16 273 CCUACGCGCGCGGCCCGGA 16 291UCCGGGCCGCGCGCGUAGG 177 291 AGCUCUGUUCCCUGGAACU 17 291AGCUCUGUUCCCUGGAACU 17 309 AGUUCCAGGGAACAGAGCU 178 309UUUGGGCACUGCCUCUGGG 18 309 UUUGGGCACUGCCUCUGGG 18 327CCCAGAGGCAGUGCCCAAA 179 327 GACCCCUGCCGGCCAGCAG 19 327GACCCCUGCCGGCCAGCAG 19 345 CUGCUGGCCGGCAGGGGUC 180 345GGCAGGAUGGUGCUUGCCU 20 345 GGCAGGAUGGUGCUUGCCU 20 363AGGCAAGCACCAUCCUGCC 181 363 UCGUGCCCCUUGGUGCCCG 21 363UCGUGCCCCUUGGUGCCCG 21 381 CGGGCACCAAGGGGCACGA 182 381GUCUGCUGAUGUGCCCAGC 22 381 GUCUGCUGAUGUGCCCAGC 22 399GCUGGGCACAUCAGCAGAC 183 399 CCUGUGCCCGCCAUGCCGC 23 399CCUGUGCCCGCCAUGCCGC 23 417 GCGGCAUGGCGGGCACAGG 184 417CCCUCCAUCUCAGCUUUCC 24 417 CCCUCCAUCUCAGCUUUCC 24 435GGAAAGCUGAGAUGGAGGG 185 435 CAGGCCGCCUACAUCGGCA 25 435CAGGCCGCCUACAUCGGCA 25 453 UGCCGAUGUAGGCGGCCUG 186 453AUCGAGGUGCUCAUCGCCC 26 453 AUCGAGGUGCUCAUCGCCC 26 471GGGCGAUGAGCACCUCGAU 187 471 CUGGUCUCUGUGCCCGGGA 27 471CUGGUCUCUGUGCCCGGGA 27 489 UCCCGGGCACAGAGACCAG 188 489AACGUGCUGGUGAUCUGGG 28 489 AACGUGCUGGUGAUCUGGG 28 507CCCAGAUCACCAGCACGUU 189 507 GCGGUGAAGGUGAACCAGG 29 507GCGGUGAAGGUGAACCAGG 29 525 CCUGGUUCACCUUCACCGC 190 525GCGCUGCGGGAUGCCACCU 30 525 GCGCUGCGGGAUGCCACCU 30 543AGGUGGCAUCCCGCAGCGC 191 543 UUCUGCUUCAUCGUGUCGC 31 543UUCUGCUUCAUCGUGUCGC 31 561 GCGACACGAUGAAGCAGAA 192 561CUGGCGGUGGCUGAUGUGG 32 561 CUGGCGGUGGCUGAUGUGG 32 579CCACAUCAGCCACCGCCAG 193 579 GCCGUGGGUGCCCUGGUCA 33 579 GCCGUGGGUGCCCUGGUCA 33 597 UGACCAGGGCACCCACGGC 194 597AUCCCCCUCGCCAUCCUCA 34 597  AUCCCCCUCGCCAUCCUCA 34 615UGAGGAUGGCGAGGGGGAU 195 615 AUCAACAUUGGGCCACAGA 35 615AUCAACAUUGGGCCACAGA 35 633 UCUGUGGCCCAAUGUUGAU 196 633ACCUACUUCCACACCUGCC 36 633 ACCUACUUCCACACCUGCC 36 651GGCAGGUGUGGAAGUAGGU 197 651 CUCAUGGUUGCCUGUCCGG 37 651CUCAUGGUUGCCUGUCCGG 37 669 CCGGACAGGCAACCAUGAG 198 669GUCCUCAUCCUCACCCAGA 38 669  GUCCUCAUCCUCACCCAGA 38 687 UCUGGGUGAGGAUGAGGAC 199 687 AGCUCCAUCCUGGCCCUGC 39 687AGCUCCAUCCUGGCCCUGC 39 705  GCAGGGCCAGGAUGGAGCU 200 705CUGGCAAUUGCUGUGGACC 40 705  CUGGCAAUUGCUGUGGACC 40 723GGUCCACAGCAAUUGCCAG 201 723 CGCUACCUCCGGGUCAAGA 41 723 CGCUACCUCCGGGUCAAGA 41 741 UCUUGACCCGGAGGUAGCG 202 741AUCCCUCUCCGGUACAAGA 42 741 AUCCCUCUCCGGUACAAGA 42 759 UCUUGUACCGGAGAGGGAU 203 759 AUGGUGGUGACCCCCCGGA 43 759AUGGUGGUGACCCCCCGGA 43 777  UCCGGGGGGUCACCACCAU 204 777AGGGCGGCGGUGGCCAUAG 44 777  AGGGCGGCGGUGGCCAUAG 44 795CUAUGGCCACCGCCGCCCU 205 795 GCCGGCUGCUGGAUCCUCU 45 795GCCGGCUGCUGGAUCCUCU 45 813 AGAGGAUCCAGCAGCCGGC 206 813UCCUUCGUGGUGGGACUGA 46 813  UCCUUCGUGGUGGGACUGA 46 831 UCAGUCCCACCACGAAGGA 207 831 ACCCCUAUGUUUGGCUGGA 47 831ACCCCUAUGUUUGGCUGGA 47 849 UCCAGCCAAACAUAGGGGU 208 849AACAAUCUGAGUGCGGUGG 48 849  AACAAUCUGAGUGCGGUGG 48 867CCACCGCACUCAGAUUGUU 209 867 GAGCGGGCCUGGGCAGCCA 49 867GAGCGGGCCUGGGCAGCCA 49 885  UGGCUGCCCAGGCCCGCUC 210 885AACGGCAGCAUGGGGGAGC 50 885  AACGGCAGCAUGGGGGAGC 50 903GCUCCCCCAUGCUGCCGUU 211 903 CCCGUGAUCAAGUGCGAGU 51 903CCCGUGAUCAAGUGCGAGU 51 921  ACUCGCACUUGAUCACGGG 212 921UUCGAGAAGGUCAUCAGCA 52 921 UUCGAGAAGGUCAUCAGCA 52 939 UGCUGAUGACCUUCUCGAA 213 939 AUGGAGUACAUGGUCUACU 53 939 AUGGAGUACAUGGUCUACU 53 957 AGUAGACCAUGUACUCCAU 214 957UUCAACUUCUUUGUGUGGG 54 957 UUCAACUUCUUUGUGUGGG 54 975CCCACACAAAGAAGUUGAA 215 975 GUGCUGCCCCCGCUUCUCC 55 975 GUGCUGCCCCCGCUUCUCC 55 993 GGAGAAGCGGGGGCAGCAC 216 993CUCAUGGUCCUCAUCUACC 56 993 CUCAUGGUCCUCAUCUACC 56 1011GGUAGAUGAGGACCAUGAG 217 1011 CUGGAGGUCUUCUACCUAA 57 1011CUGGAGGUCUUCUACCUAA 57 1029 UUAGGUAGAAGACCUCCAG 218 1029AUCCGCAAGCAGCUCAACA 58 1029 AUCCGCAAGCAGCUCAACA 58 1047UGUUGAGCUGCUUGCGGAU 219 1047 AAGAAGGUGUCGGCCUCCU 59 1047AAGAAGGUGUCGGCCUCCU 59 1065 AGGAGGCCGACACCUUCUU 220 1065UCCGGCGACCCGCAGAAGU 60 1065 UCCGGCGACCCGCAGAAGU 60 1083ACUUCUGCGGGUCGCCGGA 221 1083 UACUAUGGGAAGGAGCUGA 61 1083UACUAUGGGAAGGAGCUGA 61 1101 UCAGCUCCUUCCCAUAGUA 222 1101AAGAUCGCCAAGUCGCUGG 62 1101 AAGAUCGCCAAGUCGCUGG 62 1119CCAGCGACUUGGCGAUCUU 223 1119 GCCCUCAUCCUCUUCCUCU 63 1119GCCCUCAUCCUCUUCCUCU 63 1137 AGAGGAAGAGGAUGAGGGC 224 1137UUUGCCCUCAGCUGGCUGC 64 1137 UUUGCCCUCAGCUGGCUGC 64 1155GCAGCCAGCUGAGGGCAAA 225 1155 CCUUUGCACAUCCUCAACU 65 1155CCUUUGCACAUCCUCAACU 65 1173 AGUUGAGGAUGUGCAAAGG 226 1173UGCAUCACCCUCUUCUGCC 66 1173 UGCAUCACCCUCUUCUGCC 66 1191GGCAGAAGAGGGUGAUGCA 227 1191 CCGUCCUGCCACAAGCCCA 67 1191CCGUCCUGCCACAAGCCCA 67 1209 UGGGCUUGUGGCAGGACGG 228 1209AGCAUCCUUACCUACAUUG 68 1209 AGCAUCCUUACCUACAUUG 68 1227CAAUGUAGGUAAGGAUGCU 229 1227 GCCAUCUUCCUCACGCACG 69 1227GCCAUCUUCCUCACGCACG 69 1245 CGUGCGUGAGGAAGAUGGC 230 1245GGCAACUCGGCCAUGAACC 70 1245 GGCAACUCGGCCAUGAACC 70 1263GGUUCAUGGCCGAGUUGCC 231 1263 CCCAUUGUCUAUGCCUUCC 71 1263CCCAUUGUCUAUGCCUUCC 71 1281 GGAAGGCAUAGACAAUGGG 232 1281CGCAUCCAGAAGUUCCGCG 72 1281  CGCAUCCAGAAGUUCCGCG 72 1299CGCGGAACUUCUGGAUGCG 233 1299 GUCACCUUCCUUAAGAUUU 73 1299GUCACCUUCCUUAAGAUUU 73 1317 AAAUCUUAAGGAAGGUGAC 234 1317UGGAAUGACCAUUUCCGCU 74 1317 UGGAAUGACCAUUUCCGCU 74 1335AGCGGAAAUGGUCAUUCCA 235 1335 UGCCAGCCUGCACCUCCCA 75 1335 UGCCAGCCUGCACCUCCCA 75 1353 UGGGAGGUGCAGGCUGGCA 236 1353AUUGACGAGGAUCUCCCAG 76 1353 AUUGACGAGGAUCUCCCAG 76 1371CUGGGAGAUCCUCGUCAAU 237 1371 GAAGAGAGGCCUGAUGACU 77 1371GAAGAGAGGCCUGAUGACU 77 1389 AGUCAUCAGGCCUCUCUUC 238 1389UAGACCCCGCCUUCCGCUC 78 1389 UAGACCCCGCCUUCCGCUC 78 1407GAGCGGAAGGCGGGGUCUA 239 1407 CCCACCAGCCCACAUCCAG 79 1407CCCACCAGCCCACAUCCAG 79 1425 CUGGAUGUGGGCUGGUGGG 240 1425GUGGGGUCUCAGUCCAGUC 80 1425 GUGGGGUCUCAGUCCAGUC 80 1443GACUGGACUGAGACCCCAC 241 1443 CCUCACAUGCCCGCUGUCC 81 1443CCUCACAUGCCCGCUGUCC 81 1461 GGACAGCGGGCAUGUGAGG 242 1461CCAGGGGUCUCCCUGAGCC 82 1461  CCAGGGGUCUCCCUGAGCC 82 1479GGCUCAGGGAGACCCCUGG 243 1479 CUGCCCCAGCUGGGCUGUU 83 1479 CUGCCCCAGCUGGGCUGUU 83 1497 AACAGCCCAGCUGGGGCAG 244 1497UGGCUGGGGGCAUGGGGGA 84 1497  UGGCUGGGGGCAUGGGGGA 84 1515UCCCCCAUGCCCCCAGCCA 245 1515 AGGCUCUGAAGAGAUACCC 85 1515AGGCUCUGAAGAGAUACCC 85 1533 GGGUAUCUCUUCAGAGCCU 246 1533CACAGAGUGUGGUCCCUCC 86 1533 CACAGAGUGUGGUCCCUCC 86 1551GGAGGGACCACACUCUGUG 247 1551 CACUAGGAGUUAACUACCC 87 1551CACUAGGAGUUAACUACCC 87 1569 GGGUAGUUAACUCCUAGUG 248 1569CUACACCUCUGGGCCCUGC 88 1569 CUACACCUCUGGGCCCUGC 88 1587GCAGGGCCCAGAGGUGUAG 249 1587 CAGGAGGCCUGGGAGGGCA 89 1587CAGGAGGCCUGGGAGGGCA 89 1605 UGCCCUCCCAGGCCUCCUG 250 1605AAGGGUCCUACGGAGGGAC 90 1605 AAGGGUCCUACGGAGGGAC 90 1623GUCCCUCCGUAGGACCCUU 251 1623 CCAGGUGUCUAGAGGCAAC 91 1623CCAGGUGUCUAGAGGCAAC 91 1641 GUUGCCUCUAGACACCUGG 252 1641CAGUGUUCUGAGCCCCCAC 92 1641 CAGUGUUCUGAGCCCCCAC 92 1659GUGGGGGCUCAGAACACUG 253 1659 CCUGCCUGACCAUCCCAUG 93 1659CCUGCCUGACCAUCCCAUG 93 1677 CAUGGGAUGGUCAGGCAGG 254 1677GAGCAGUCCAGCGCUUCAG 94 1677  GAGCAGUCCAGCGCUUCAG 94 1695CUGAAGCGCUGGACUGCUC 255 1695 GGGCUGGGCAGGUCCUGGG 95 1695GGGCUGGGCAGGUCCUGGG  95 1713 CCCAGGACCUGCCCAGCCC 256 1713GGAGGCUGAGACUGCAGAG 96 1713 GGAGGCUGAGACUGCAGAG 96 1731CUCUGCAGUCUCAGCCUCC 257 1731 GGAGCCACCUGGGCUGGGA 97 1731GGAGCCACCUGGGCUGGGA 97 1749 UCCCAGCCCAGGUGGCUCC 258 1749AGAAGGUGCUUGGGCUUCU 98 1749 AGAAGGUGCUUGGGCUUCU 98 1767AGAAGCCCAAGCACCUUCU 259 1767 UGCGGUGAGGCAGGGGAGU 99 1767 UGCGGUGAGGCAGGGGAGU 99 1785 ACUCCCCUGCCUCACCGCA 260 1785UCUGCUUGUCUUAGAUGUU 100 1785 UCUGCUUGUCUUAGAUGUU 100 1803AACAUCUAAGACAAGCAGA 261 1803 UGGUGGUGCAGCCCCAGGA 101 1803UGGUGGUGCAGCCCCAGGA 101 1821 UCCUGGGGCUGCACCACCA 262 1821ACCAAGCUUAAGGAGAGGA 102 1821  ACCAAGCUUAAGGAGAGGA 102 1389UCCUCUCCUUAAGCUUGGU 263 1839 AGAGCAUCUGCUCUGAGAC 103 1839 AGAGCAUCUGCUCUGAGAC 103 1957 GUCUCAGAGCAGAUGCUCU 264 1857CGGAUGGAAGGAGAGAGGU 104 1857 CGGAUGGAAGGAGAGAGGU 104 1875ACCUCUCUCCUUCCAUCCG 265 1875 UUGAGGAUGCACUGGCCUG 105 1875UUGAGGAUGCACUGGCCUG  105 1893 CAGGCCAGUGCAUCCUCAA 266 1893GUUCUGUAGGAGAGACUGG 106 1893 GUUCUGUAGGAGAGACUGG  106 1911CCAGUCUCUCCUACAGAAC 267 1911 GCCAGAGGCAGCUAAGGGG 107 1911GCCAGAGGCAGCUAAGGGG 107 1929 CCCCUUAGCUGCCUCUGGC  268 1929GCAGGAAUCAAGGAGCCUC 108 1929 GCAGGAAUCAAGGAGCCUC  108 1947GAGGCUCCUUGAUUCCUGC 269 1947 CCGUUCCCACCUCUGAGGA 109 1947CCGUUCCCACCUCUGAGGA  109 1965 UCCUCAGAGGUGGGAACGG 270 1965ACUCUGGACCCCAGGCCAU 110 1965 ACUCUGGACCCCAGGCCAU 110 1983AUGGCCUGGGGUCCAGAGU 271 1983 UACCAGGUGCUAGGGUGCC 111 1983UACCAGGUGCUAGGGUGCC  111 2001 GGCACCCUAGCACCUGGUA  272 2001CUGCUCUCCUUGCCCUGGG 112 2001 CUGCUCUCCUUGCCCUGGG  112 2019CCCAGGGCAAGGAGAGCAG  273 2019 GCCAGCCCAGGAUUGUACG 113 2019GCCAGCCCAGGAUUGUACG  113 2037 CGUACAAUCCUGGGCUGGC  274 2037GUGGGAGAGGCAGAAAGGG 114 2037 GUGGGAGAGGCAGAAAGGG 114 2055CCCUUUCUGCCUCUCCCAC  275 2055 GUAGGUUCAGUAAUCAUUU 115 2055GUAGGUUCAGUAAUCAUUU  115 2073 AAAUGAUUACUGAACCUAC 276 2073UCUGAUGAUUUGCUGGAGU 116 2073 UCUGAUGAUUUGCUGGAGU  116 2091ACUCCAGCAAAUCAUCAGA  277 2091 UGCUGGCUCCACGCCCUGG 117 2091UGCUGGCUCCACGCCCUGG  117 2109 CCAGGGCGUGGAGCCAGCA  278 2109GGGAGUGAGCUUGGUGCGG 118 2109 GGGAGUGAGCUUGGUGCGG  118 2127CCGCACCAAGCUCACUCCC  279 2127 GUAGGUGCUGGCCUCAAAC 119 2127GUAGGUGCUGGCCUCAAAC  119 2145 GUUUGAGGCCAGCACCUAC  280 2145CAGCCACGAGGUGGUAGCU 120 2145 CAGCCACGAGGUGGUAGCU  120 2163AGCUACCACCUCGUGGCUG  281 2163 UCUGAGCCCUCCUUCUUGC 121 2163UCUGAGCCCUCCUUCUUGC 121 2181 GCAAGAAGGAGGGCUCAGA  282 2181CCCUGAGCUUUCCGGGGAG 122 2181 CCCUGAGCUUUCCGGGGAG 122 2199CUCCCCGGAAAGCUCAGGG 283 2199 GGAGCCUGGAGUGUAAUUA 123 2199GGAGCCUGGAGUGUAAUUA  123 2217 UAAUUACACUCCAGGCUCC  284 2217ACCUGUCAUCUGGGCCACC 124 2217 ACCUGUCAUCUGGGCCACC  124 2235GGUGGCCCAGAUGACAGGU 285 2235 CAGCUCCACUGGCCCCCGU 125 2235CAGCUCCACUGGCCCCCGU  125 2253 ACGGGGGCCAGUGGAGCUG  286 2253UUGCCGGGCCUGGACUGUC 126 2253 UUGCCGGGCCUGGACUGUC  126 2271GACAGUCCAGGCCCGGCAA  287 2271 CCUAGGUGACCCCAUCUCU 127 2271CCUAGGUGACCCCAUCUCU 127 2289 AGAGAUGGGGUCACCUAGG  288 2289UGCUGCUUCUGGGCCUGAU 128 2289 UGCUGCUUCUGGGCCUGAU 128 2307AUCAGGCCCAGAAGCAGCA  289 2307 UGGAGAGGAGAACACUAGA 129 2307UGGAGAGGAGAACACUAGA 129 2325 UCUAGUGUUCUCCUCUCCA  290 2325ACAUGCCAACUCGGGAGCA 130 2325 ACAUGCCAACUCGGGAGCA  130 2343UGCUCCCGAGUUGGCAUGU  291 2343 AUUCUGCCUGCCUGGGAAC 131 2343AUUCUGCCUGCCUGGGAAC  131 2361 GUUCCCAGGCAGGCAGAAU  292 2361CGGGGUGGACGAGGGAGUG 132 2361 CGGGGUGGACGAGGGAGUG  132 2379CACUCCCUCGUCCACCCCG 293 2379 GUCUGUAAGGACUCAGUGU 133 2379GUCUGUAAGGACUCAGUGU  133 2397 ACACUGAGUCCUUACAGAC 294 2397UUGACUGUAGGCGCCCCUG 134 2397 UUGACUGUAGGCGCCCCUG  134 2415CAGGGGCGCCUACAGUCAA 295 2415 GGGGUGGGUUUAGCAGGCU 135 2415GGGGUGGGUUUAGCAGGCU 135 2433 AGCCUGCUAAACCCACCCC  296 2433UGCAGCAGGCAGAGGAGGA 136 2433 UGCAGCAGGCAGAGGAGGA 136 2451UCCUCCUCUGCCUGCUGCA  297 2451 AGUACCCCCCUGAGAGCAU 137 2451AGUACCCCCCUGAGAGCAU  137 2469 AUGCUCUCAGGGGGGUACU 298 2469UGUGGGGGAAGGCCUUGCU 138 2469 UGUGGGGGAAGGCCUUGCU  138 2487AGCAAGGCCUUCCCCCACA  299 2487 UGUCAUGUGAAUCCCUCAA 139 2487UGUCAUGUGAAUCCCUCAA  139 2505 UUGAGGGAUUCACAUGACA  300 2505AUACCCCUAGUAUCUGGCU 140 2505 AUACCCCUAGUAUCUGGCU  140 2523AGCCAGAUACUAGGGGUAU  301 2523 UGGGUUUUCAGGGGCUUUG 141 2523UGGGUUUUCAGGGGCUUUG  141 2541 CAAAGCCCCUGAAAACCCA  302 2541GGAAGCUCUGUUGCAGGUG 142 2541 GGAAGCUCUGUUGCAGGUG 142 2559CACCUGCAACAGAGCUUCC 303 2559 GUCCGGGGGUCUAGGACUU 143 2559GUCCGGGGGUCUAGGACUU 143 2577 AAGUCCUAGACCCCCGGAC 304 2577UUAGGGAUCUGGGAUCUGG 144 2577 UUAGGGAUCUGGGAUCUGG 144 2595CCAGAUCCCAGAUCCCUAA 305 2595 GGGAAGGACCAACCCAUGC 145 2595GGGAAGGACCAACCCAUGC 145 2613 GCAUGGGUUGGUCCUUCCC 306 2613CCCUGCCAAGCCUGGAGCC 146 2613 CCCUGCCAAGCCUGGAGCC 146 2631GGCUCCAGGCUUGGCAGGG 307 2631 CCCUGUGUUGGGGGGCAAG 147 2631CCCUGUGUUGGGGGGCAAG 147 2649 CUUGCCCCCCAACACAGGG 308 2649GGUGGGGGAGCCUGGAGCC 148 2649 GGUGGGGGAGCCUGGAGCC 148 2667GGCUCCAGGCUCCCCCACC 309 2667 CCCUGUGUGGGAGGGCGAG 149 2667CCCUGUGUGGGAGGGCGAG 149 2685 CUCGCCCUCCCACACAGGG 310 2685GGCGGGGGAGCCUGGAGCC 150 2685 GGCGGGGGAGCCUGGAGCC 150 2703GGCUCCAGGCUCCCCCGCC 311 2703 CCCUGUGUGGGAGGGCGAG 151 2703CCCUGUGUGGGAGGGCGAG 151 2721 CUCGCCCUCCCACACAGGG 312 2721GGCGGGGGAUCCUGGAGCC 152 2721 GGCGGGGGAUCCUGGAGCC 152 2739GGCUCCAGGAUCCCCCGCC 313 2739 CCCUGUGUCGGGGGGCGAG 153 2739CCCUGUGUCGGGGGGCGAG 153 2757 CUCGCCCCCCGACACAGGG 314 2757GGGAGGGGAGGUGGCCGUC 154 2757 GGGAGGGGAGGUGGCCGUC 154 2775GACGGCCACCUCCCCUCCC 315 2775 CGGUUGACCUUCUGAACAU 155 2775CGGUUGACCUUCUGAACAU 155 2793 AUGUUCAGAAGGUCAACCG 316 2793UGAGUGUCAACUCCAGGAC 156 2793 UGAGUGUCAACUCCAGGAC 156 2811GUCCUGGAGUUGACACUCA 317 2811 CUUGCUUCCAAGCCCUUCC 157 2811CUUGCUUCCAAGCCCUUCC 157 2829 GGAAGGGCUUGGAAGCAAG 318 2829CCUCUGUUGGAAAUUGGGU 158 2829 CCUCUGUUGGAAAUUGGGU 158 2847ACCCAAUUUCCAACAGAGG 319 2847 UGUGCCCUGGCUCCCAAGG 159 2847UGUGCCCUGGCUCCCAAGG 159 2865 CCUUGGGAGCCAGGGCACA 320 2865GGAGGCCCAUGUGACUAAU 160 2865 GGAGGCCCAUGUGACUAAU 160 2883AUUAGUCACAUGGGCCUCC 321 2880 UAAUAAAAAACUGUGAACC 161 2880UAAUAAAAAACUGUGAACC 161 2898 GGUUCACAGUUUUUUAUUA 322 NM_000674|ADORA1The 3′-ends of the Upper sequence and the Lower sequence of the siRNAconstruct can include a overhang sequence, for example about 1, 2, 3, or4 nucleotides in length, preferably 2 nucleotides in length, wherein theoverhanging sequence of the lower sequence is optionally complementaryto a portion of the target sequence. The upper sequence is also referredto as the sense strand, whereas the lower sequence is also referred toas the antisense strand.

TABLE II Wait Time* Wait Time* Wait Time* Reagent Equivalents Amount DNA2′-O-methyl RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5sec 5 sec N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 μL 45sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 secAcetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124 μL 5 sec5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 sec Iodine 20.6244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300 sec 300 secAcetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96 wellInstrument Equivalents: DNA/2′- Amount: DNA/2′-O- Wait Time* Wait Time*Wait Time* Reagent O-methyl/Ribo methyl/Ribo DNA 2′-O-methyl RiboPhosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 sec S-EthylTetrazole 70/105/210 40/60/120 μL 60 sec 180 min 360 sec AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does not includecontact time during delivery. Tandem synthesis utilizes double couplingof linker molecule

TABLE III Chemically Modified siRNAs Target Pos Aliases Sequence (5'-3')Seq ID Strand 1819 ADORA1: 1821U21 siRNA stab4 B AccAAGcuuAAGGAGAGGAGA B340 Upper  919 ADORA1: 921U21 siRNA stab4 B uucGAGAAGGucAucAGcAuG B 342Upper 1621 ADORA1: 1623U21 siRNA stab4 B ccAGGuGucuAGAGGcAAcAG B 344Upper 2773 ADORA1: 2775U21 siRNA stab4 B cGGuuGAccuucuGAAcAuGA B 346Upper 1819 ADORA1: 1839L21 siRNA (1821C) stab5 uccucuccuuAAGcuuGGuTsT341 Lower  919 ADORA1: 939L21 siRNA (921C) stab5 uGcuGAuGAccuucucGAATsT343 Lower 1621 ADORA1: 1641L21 siRNA (1623C) stab5GuuGccucuAGAcAccuGGTsT 345 Lower 2773ADORA1: 2793L21 siRNA (2775C) stab5 AuGuucAGAAGGucAAccGTsT 348 LowerUppercase = 2′-OH u, c = 2′-fluoro U, C T = deoxy T B = inverted deoxyabasic s = phosphorothioate linkage

1-20. (canceled)
 21. A chemically modified short interfering ribonucleic acid (siRNA) molecule, wherein: (a) the siRNA molecule comprises a sense strand and a separate antisense strand; (b) each strand of the siRNA is independently 18 to 24 nucleotides in length, and the siRNA comprises between 17 and 23 base pairs; (c) one strand of the siRNA is complementary to a human ADORA1 RNA sequence; (d) 10 or more pyrimidine nucleotides of the sense and antisense strand are 2′-deoxy, 2′-O-methyl, or 2′-deoxy-2′-fluoro pyrimidine nucleotides; (e) the siRNA molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphorothioate internucleotide linkages; and, (f) the sense strand of the siRNA molecule comprises a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends.
 22. The siRNA molecule of claim 21, wherein one or more of the pyrimidine nucleotides in the sense strand are 2′-O-methyl pyrimidine nucleotides.
 23. The siRNA molecule of claim 22, wherein all of the pyrimidine nucleotides in the sense strand are 2′-O-methyl pyrimidine nucleotides.
 24. The siRNA molecule of claim 22, wherein one or more of the pyrimidine nucleotides in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides.
 25. The siRNA molecule of claim 24, wherein all of the pyrimidine nucleotides in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides.
 26. The siRNA molecule of claim 21, wherein one or more of the pyrimidine nucleotides in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides.
 27. The siRNA molecule of claim 26, wherein all of the pyrimidine nucleotides in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides.
 28. The siRNA molecule of claim 21, wherein the terminal cap is an inverted deoxy abasic moiety.
 29. The siRNA molecule of claim 21, wherein one or more of the pyrimidine nucleotides in the sense stand are 2′-deoxy-2′-fluoro pyrimidine nucleotides.
 30. The siRNA molecule of claim 29, wherein all of the pyrimidine nucleotides in the sense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides.
 31. The siRNA molecule of claim 21, wherein both the sense strand and the antisense strand include a 3′-overhang of two nucleotides.
 32. The siRNA molecule of claim 31, wherein the nucleotides of the 3′-overhangs are 2′-deoxy nucleotides.
 33. The siRNA molecule of claim 21, wherein the antisense strand has a phosphorothioate internucleotide linkage at the 3′ end.
 34. A composition comprising the siRNA molecule of claim 21 in a pharmaceutically acceptable carrier or diluent. 